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Abstract: Estrogen receptors (ERs) are steroid hormone receptors, which belong to a large nuclear receptor family. Endocrine diseases correlate strongly with dysregulated ER signaling. Traditional therapies continue to rely on small molecule inhibitors, including aromatase inhibitors (AIs) and selective estrogen receptor modulators (SERMs), all of which permit acquired resistance to endocrine therapy. Proteolytic targeting chimeras (PROTACs) offer unprecedented potential for solving acquired endocrine resistance. ARV-471, an ER-targeting PROTAC developed by Arvinas, was approved by the US FDA to entered clinical trials in 2019 to treat patients suffering from locally advanced or metastatic ER-positive/HER2-negative breast cancer, and has since been approved by the US FDA. In this review, we will focus on progress in developing ER-targeting PROTACs from publications and patents aimed at the treatment of endocrine diseases.

Endometriosis is defined as the presence of endometrial glands and stroma outside the uterus. Adolescent endometriosis is often confined to the pelvic cavity and is a common cause of secondary dysmenorrhea in adolescents. Adolescent endometriosis is often with delayed diagnosis. Early diagnosis and intervention can prevent the damage of pelvic structure and ovarian tissue and reduce the incidence of adhesion and infertility. Adolescent endometriosis can be diagnosed and treated by laparoscopic biopsy. There are many kinds of drugs to treat endometriosis, such as NSAIDs, progesterone, selective progesterone receptor antagonists, GnRH-a, gonadotropin-releasing hormone antagonists, aromatase inhibitors, dopamine agonists, angiogenesis inhibitors, vegetable drugs, traditional Chinese medicine prescriptions. Future treatment options, including Future treatment options include CTZ, vitamin D3, oxytocin receptor inhibitors, melatonin, doxycycline, bevacizumab, curcumin, isotretinoin, and rosiglitazone, etc can inhibit ectopic lesions. This article mainly summarizes advances in medical treatment underlying adolescent endometriosis, and provides guidance for the early clinical diagnosis and intervention of adolescent endometriosis, so as to improve the quality of life of patients and reduce adverse outcomes.

Background: Many natural and synthetic flavonoids have been studied and documented by inhibiting aromatase enzymes for their anti-cancer activity against breast carcinoma. The aromatase enzyme is a possible target for the estrogen's positive breast cancer receptor. Objective: Hence, a series of flavonoids have been synthesized and assessed for their in vitro cytotoxicity and aromatase inhibitory activity. Methods: 39 Flavonoids were synthesized and characterized by spectroscopic techniques, and their computational study was performed using the maestro version of the Schrodinger. In-silico ADME properties were checked by QikProp software. A total of 18 compounds were evaluated based on the docking score using cytotoxicity assay in human breast cancer cell line MCF-7. Results: Of the 18 compounds tested, 07 compounds, namely 2b, 8b, 14b, 15b, 19b, 24b, and 30b flavonoids were found to be more active with their IC50 values of 20.73 μM, 1.636 μM, 16.08 μM, 22.02 μM, 15.75 μM, 0.345 μM and 16.08 μM, respectively, compared with the reference drug letrozole. The in-vitro aromatase inhibitory activity of six compounds 2b, 8b, 14b, 19b, 24b, and 30b was conducted using a fluorogenic assay kit. The values of IC50 for compounds 2b and 24b were found to be 0.31 μM and 0.36 μM, respectively. Conclusion: Therefore, it was concluded that compounds 2b and 24b had a potent inhibitory effect of aromatase compared with letrozole with an IC50 value of 0.86 μM. At the same time, the other compounds 8b, 14b, 30b, and 19b were considered to have similar aromatase inhibitory activity. Hence, their essential aromatase inhibitory activities make them good lead candidates for developing potent inhibitors of aromatase.

Chapter 3 discusses cancer chemotherapy related to the hormonal regulation of tumors, starting with the regulation of gene transcription by estrogens and the proposed mechanisms to explain the development of estrogen-dependent tumors. Nonsteroidal antiestrogens, also known as “selective estrogen receptor modulators” (SERMs), are explained at the molecular and atomic levels, and their action is rationalized by using the structures of their complexes with the receptor. The mechanisms of resistance to hormonal therapy are discussed, as well as alternative endocrine treatments. After explaining different mechanisms to rationalize aromatase action, steroidal (type I) and nonsteroidal (type II) aromatase inhibitors are studied, first discussing the advantages of the mechanism-based inhibitors known as aromatase inactivators and then the structure–activity relationships in steroidal aromatase inhibitors, especially how the coordination of azole derivatives to the iron atom of the heme group of the enzyme explains their affinity but also their problematic selectivity. We comment on the role of steroid sulfatase in steroid biosynthesis, the mechanism of action of its inhibitors, and the advantages of dual aromatase–sulfatase inhibitors. The chapter discusses the importance in prostatic cancer of steroidal and nonsteroidal antiandrogens and the resistance associated with the presence of a variant of the androgen receptors, as well as inhibitors of the different enzymes involved in androgen biosynthesis. The mechanisms associated with the anticancer activity of agonists of the gestagen receptor, glucocorticoids, retinoids, and agonists of vitamin D receptors and peroxysome proliferator activating receptors (PPARγ) are also commented.

Abstract: Numerous novel medicinal agents isolated from plant sources were used as indigenous remedies for the management and treatment of various types of cancer diseases. Naringenin is a naturally occurring flavanone glycoside, and aglycone (genin) moiety of naringin, predominantly found in citrus and grapefruits, has emerged as a potential therapeutic agent for the management of a variety of diseases. A number of scientific papers have been published on naringenin describing its detailed studies and its therapeutic application in different diseases. The current study highlights a comprehensive study on naringenin concerning its biosynthesis, molecular targets/pathways involved in carcinogenesis, mechanism of actions (MOAs), and structure-activity relationships (SARs), and patents granted have been highlighted. Naringenin and its derivatives have great anti-cancer activity due to their inhibitory potential against diverse targets, namely ABCG2/P-gp/BCRP, 5a-reductase, 17- bhydroxysteroid dehydrogenase, aromatase, proteasome, HDAC/Situin-1, VEGF, VEGFR-2 kinase, MMP-2/9, JAK/STAT signaling pathways, CDC25B, tubulin, topoisomerase-II, cathepsin-K, Wnt, NF-kB, B-Raf and mTOR, etc. With the in-depth knowledge of molecular targets, structural intuition, and SARs, the current study may be beneficial to design more potent, safe, effective, and economic anti-cancer naringenin. This has been concluded that naringenin is a promising natural product for the management and therapy of cancer. Further evolution for pharmacological importance, clinical research, and trials are required to manifest its therapeutic action on metabolic syndrome in the human community.

Vitamin D is used to reduce cancer risk and improve the outcome of cancer patients, but the vitamin D receptor (VDR; also known as the calcitriol receptor) pathway needs to be functionally intact to ensure the biological effects of circulating calcitriol, the active form of vitamin D. Besides estrogen receptor alpha (ERα), estrogen-related receptor alpha (ERRα) has also been shown to interfere with the VDR pathway, but its role in the antitumor and transactivation activity of calcitriol is completely unknown in breast cancer (BC). We observed that ERRα functionally supported the proliferation of BC cell lines and acted as a calcitriol-induced regulator of VDR. As such, ERRα deregulated the calcitriol-VDR transcription by enhancing the expression of CYP24A1 as well as of both ERα and aromatase (CYP19A1) in calcitriol-treated cells. ERRα knockdown limited the effect of calcitriol by reducing calcitriol-induced G0/G1 phase cell cycle arrest and by affecting the expression of cyclin D1 and p21/Waf. The interactome analysis suggested that Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-α (PGC-1α) and Proline-, glutamic acid-, and leucine-rich protein 1 (PELP-1) are key players in the genomic actions of the calcitriol-VDR-ERRα axis. Evaluation of patient outcomes in the The Cancer Genome Atlas (TCGA) dataset showed the translational significance of the biological effects of the VDR-ERRα axis, highlighting that VDR, CYP24A1 and ERRα overexpression correlates with poor prognosis in basal-like BC.

The preservation of fertility in women of childbearing age with breast cancer is challenging because the time for ovarian stimulation is restricted and only a limited number of oocytes can be retrieved before gonadotoxic therapies. The aim of this meta-analysis was to evaluate the fertility preservation outcomes after ovarian stimulation with various protocols in women with breast cancer. PubMed, Embase and the Cochrane Library were searched. Twenty-two studies comparing the outcomes of women with breast cancer receiving random-start ovarian stimulation or conventional protocol; single or double ovarian stimulation cycles; and coadministration of aromatase inhibitors or tamoxifen were included. Random-start ovarian stimulation resulted in a comparable number of retrieved oocytes to the conventional protocol. Two ovarian stimulation cycles had significantly higher numbers of total retrieved oocytes than one cycle (mean difference 7.91, 95% confidence interval [CI] 3.42 to 12.40). Coadministration of letrozole and tamoxifen showed similar results for retrieved oocytes to those without. A significantly lower peak serum oestradiol concentration was observed in letrozole-based groups than in letrozole-free groups (mean difference -1.22; 95% CI -1.42 to -1.02). In conclusion, this study indicated that implementing random-start protocols to shorten the duration of waiting for ovarian stimulation, applying two ovarian stimulation cycles, and coadministration of letrozole can lead to more desirable outcomes.

527 Background: Steroidal and non-steroidal aromatase inhibitors (AIs) provide better therapeutic response in comparison to other endocrine therapies in the adjuvant treatment of breast cancer (BC). They interfere with bone turnover which may lead to increased incidence of bone related safety events. There are no head-on studies comparing the incidence of bone safety events and hence we performed this network meta-analysis (NMA) to compare the incidence of bone safety events among patients treated with steroidal and non-steroidal AIs. Methods: Literature searches were performed in PubMed and Embase with key words to identify randomized controlled trials in BC patients treated with AIs in adjuvant settings compared against tamoxifen or other AIs, reporting any bone-related safety events. The study was designed as per PRISMA guidelines; publication bias was evaluated by comparison-adjusted funnel plots. Bayesian NMA was done by R software (ver 3.2), GemtC package. Odds ratio (OR) and the surface under the cumulative ranking curve (SUCRA) values were used to interpret the results. Results: 15 studies reporting 6 different bone-related endpoints were included. Treatment with steroidal AI, exemestane led to lower incidence of bone pain (OR Vs anastrozole and letrozole: 0.59, p=0.63; 0.54, p=0.75), fracture episodes (OR Vs anastrozole and letrozole: 0.84, p=0.41; 0.85, p=0.73), joint stiffness (OR Vs anastrozole: 0.55, p=0.73) and osteoporosis (OR Vs anastrozole and letrozole: 0.86, p=0.41; 0.74, p=0.29) (Table) in comparison to letrozole and anastrozole. Reduction in bone mineral density was also lesser in exemestane than anastrozole (mean reduction in hip: 1.08; lumbar spine: 1.34). SUCRA values suggested exemestane to be the drug with maximum likelihood for reducing the incidence of bone-related adverse events. Conclusions: This NMA suggested that bone-related safety events might be lower in early BC patients treated with exemestane in comparison to non-steroidal AIs, anastrozole and letrozole. Although there was no statistical significance, further head-on studies are required to substantiate our results.[Table: see text]

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, while the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene and when it is cleaved at the C-terminal end, asprosin is produced. Asprosin is associated with various metabolic parameters and sex-related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth in vivo. In Exp. 1, ovaries were collected from a local slaughterhouse, follicular fluid and granulosa cells from small (6 mm) and large (6-13 mm) follicles were aspirated, cellular RNA extracted for gene expression analysis, data analyzed using ANOVA, and Pearson correlation coefficients were calculated among FBN1, OR4M1, and CYP19A1 gene expression. In Exp. 2, an intra-follicular injection of asprosin (600 ng of asprosin/194 μL of PBS) or vehicle (200 μL of PBS; Controls) was given via the theca layer of the dominant follicle of synchronized cows (n = 5/group) 1 day after injection of PGF2α, follicle sizes were measured daily via transrectal ultrasonography for 3 days, a two-way repeated measures ANOVA was used to determine the effect of asprosin on growth rate of follicles from day 0-2, and Chi-square analysis for the percentage of cows ovulated 2 days following asprosin injections. In Exp. 1, FBN1 mRNA abundance was 1.9-fold greater in cells of follicular aspirates from small than large follicles (P 0.05), but abundance of OR4M1 and CYP19A1 mRNA did not differ (P 0.10) between the two sizes of follicles. Abundance of FBN1 mRNA was positively correlated with CYP19A1 (r = 0.55, P 0.05) and OR4M1 mRNA (r = 0.50, P 0.06) across follicle sizes. In Exp. 2, cows treated with asprosin revealed a greater follicle growth rate from day 0-2 (63.4% increase in diameter) than placebo cows (36.8% increase in diameter) post-injection, and more follicles from asprosin treatment vs. control group (100% vs. 20%; P 0.05) ovulated within 2 days. These findings suggest that FBN1 may be developmentally regulated in follicular cells, and that asprosin may induce follicular growth in buffaloes, but further studies will be required to determine if asprosin directly regulates estradiol production during follicle development.

In antral follicles, the transition of proliferative granulosa cells to estradiol-producing is critical for proper oocyte maturation. MicroRNAs are noncoding RNAs that play important roles in ovarian follicular development; however, this has yet to be fully characterized. MicroRNA-21 is significantly higher in granulosa cells isolated from large antral follicles compared to those from small antral follicles. To investigate the function of miR-21, porcine granulosa cells were transfected with miR-21 mimic or miR-21 targeted siRNA. Cells with the miR-21 mimic had higher aromatase expression and estradiol production but decreased WT1 expression. Conversely, cells with the miR-21 siRNA secreted less estradiol and had higher WT1 expression. We hypothesized that miR-21 promotes estradiol production by inhibiting WT1 protein synthesis. We found a potential miR-21 binding site in the 3’UTR of the WT1 transcript and performed a dual-luciferase reporter assay using the WT and mutated 3’UTR. Compared to the negative control, the miR-21 mimic induced a significant decrease in luciferase activity in the WT 3’UTR. This decrease was reversed when the 3’UTR was mutated, suggesting miR-21 targets this site to inhibit WT1 expression. We next transfected porcine granulosa cells with WT1 targeted siRNA and observed a significant increase in aromatase expression and estradiol secretion. We propose that miR-21 represses WT1 expression in granulosa cells to potentially promote aromatase expression and estradiol production. This study offers the first report of a microRNA regulating WT1 expression in granulosa cells and reveals the role of miR-21 in WT1’s regulation of estradiol production.

Summary After the discovery of ERβ, a novel role for dihydrotestosterone (DHT) in estrogen signaling was revealed. Instead of just being a better androgen, DHT was found to be a precursor of the ERβ agonist 5α-androstane-3β, 17β-diol (3βAdiol), an estrogen which does not require aromatase for its synthesis. ERβ was found to oppose androgen signaling and thus is a potential target for treatment of prostate cancer. ERβ was also found to have effects that were independent of androgen signaling, particularly in the CNS. Although in rodent models of neurodegenerative diseases (Parkinson’s disease, multiple sclerosis, and Alzheimer’s disease), ERβ agonists are very effective in relieving symptoms and improving pathologies, this has not proven to be the case in humans. In this review we will focus on the main differences in ERβ signaling between rodents and humans and will make the point that a very important difference between the two species is in the splice variants which are expressed in humans and not rodents. The main conclusion at this point is that before we think of using ERβ agonists clinically, much more work on ERβ signaling in the human or in primates needs to be done.

Flavonoids have been shown to target aromatase, suppressing the transformed cells' proliferation and growth. Such experimental data further promoted the usage of flavonoids as an aromatase inhibitor and helps prevent cancer, specifically breast and lung cancer. Conversely, flavonoids have certain limitations like low absorption, potency, and some side effects in addition to their tremendous advantages. The pharmacokinetics and toxicity of flavonoids are now being addressed by using advanced nanotechnological approaches. This review discusses the comprehensive aromatase signaling pathway in normal and cancer cells. It also draws attention to how do flavonoids modulate aromatase signaling pathways. Also, different flavonoid groups inhibiting aromatase activities are discussed and listed in the Table comprising flavonoids group, cancer type, clinical trials, IC50, and the assay employed. Moreover, nanoparticles-mediated improvement in the pharmacokinetics and toxicity issues of flavonoids in targeting aromatase are also deliberated. In conclusion, flavonoids act as a potential anticancer agent via targeting aromatase. Besides, nanotechnological approaches are useful in addressing the pharmacokinetics and toxicity of flavonoids.

As of January 2021, there are more than 3.8 million women in the United States with a history of breast cancer. The current standard of care for breast cancer involves surgical resection, radiation therapy, adjuvant endocrine therapy, and/or adjuvant chemotherapy. Aromatase inhibitors (AIs) are the gold standard for endocrine therapy in postmenopausal women. Dermatologic adverse events (dAEs) associated with AIs are rare but have been reported in the literature. Commonly reported dAEs include unspecified rash, pruritus, alopecia, vulvovaginal atrophy, vasculitis, and autoimmune/connective tissue disorders. Appropriate preventative strategies and careful management considerations have the potential to optimize the comprehensive care of patients with cancer and improve quality of life. Furthermore, prevention of dAEs can lead to a reduction in cancer treatment interruptions and discontinuations. Herein, we characterize dAEs of AIs and discuss preventative management to reduce the incidence of AI therapy interruption.

Abstract: Breast cancer is the most frequent diagnosed cancer in women and the second most common form of cancer, causing death after lung cancer, all across the globe at an alarming rate. The level of estrogens, in breast cancer tissues of postmenopausal women is 10-40 folds higher than the non-carcinogenic breast tissues. As a result of this greater level of estrogen, breast tissue becomes more prone to develop breast cancer; mainly estradiol plays a significant role in the initiation and development of hormone dependent breast cancer. Androstenedione, Adrenal dehydroepiandrosterone sulfate and estrone-sulfate also plays an important role of precursor for estrogen biosynthesis. Estrogens deprivation exhibits an attractive phenomena in the advancement of most ideal therapeutics for the treatment of breast cancer. Inhibition of aromatase and sulphatase emerged as attractive therapy for the treatment of hormone dependent breast cancer via deprivation of estrogen by different pathways. The cocktail of aromatase and sulphatase inhibitors known as dual aromatase-sulphatase inhibitors (DASIs) emerged as an attractive approach for the effective estrogen deprivation. The present review article focused on the journey of dual aromatase-sulphatase inhibitors from the beginning to till date (2020). Keeping in view the key observations, this review may be helpful for medicinal chemists to design and develop new and efficient dual aromatase-sulphatase inhibitors for the possible treatment of hormone-related breast cancer.

Background Whereas the genomic landscape of endocrine‐resistant breast cancer has been intensely characterized in previously treated cases with local or distant recurrence, comparably little is known about genomic alterations conveying primary non‐responsiveness to endocrine treatment in luminal early breast cancer. Methods In this study, 622 estrogen receptor‐expressing breast cancer cases treated with short‐term preoperative endocrine therapy (pET) from the WSG‐ADAPT trial (NCT01779206) were analyzed for genetic alterations associated with impaired endocrine proliferative response (EPR) to 3‐week pET with tamoxifen or aromatase inhibitors. EPR was categorized as optimal (post‐pET Ki67, Early recognition of endocrine resistance could be enabled by impaired suppression of proliferation after short‐term preoperative endocrine therapy (pET). Impaired endocrine proliferative response (EPR, post‐pET Ki67 ≥10%) sorts out cancers carrying TP53 mutations.

Many breast cancer patients harbor high estrogen receptor (ER) expression in tumors that can be treated with endocrine therapy, which includes aromatase inhibitors (AI); unfortunately, resistance often occurs. Mitochondrial dysfunction has been thought to contribute to progression and to be related to hormone receptor expression in breast tumors. Mitochondrial alterations in AI-resistant breast cancer have not yet been defined. In this study, we characterized mitochondrial alterations and their roles in AI resistance. MCF-7aro AI-resistant breast cancer cells were shown to have significant changes in mitochondria. Low expressions of mitochondrial genes and proteins could be poor prognostic factors for breast cancer patients. Long-term mitochondrial inhibitor treatments-mediated mitochondrial stress adaptation could induce letrozole resistance. ERα-amphiregulin (AREG) loop signaling was activated and contributed to mitochondrial stress adaptation-mediated letrozole resistance. The up-regulation of AREG-epidermal growth factor receptor (EGFR) crosstalk activated the PI3K/Akt/mTOR and ERK pathways and was responsible for ERα activation. Moreover, mitochondrial stress adaptation-increased intracellular levels of reactive oxygen species (ROS) and calcium were shown to induce AREG expression and secretion. In conclusion, our results support the claim that mitochondrial stress adaptation contributes to AI resistance via ROS/calcium-mediated AREG-ERα loop signaling and provide possible treatment targets for overcoming AI resistance.

Women with hormone receptor positive breast cancer may receive 5 years of treatment with aromatase inhibitors but the magnitude of benefit was relatively small. Our goal was to develop a tool for identification of women with limited treatment benefit. Regression analyses were applied to women treated by placebo in CCTG MA.17R trial (NCT00754845) to identify important prognostic factors associated with distant recurrence and develop a nomogram for predicting 5-year likelihood of distant recurrence, which was internally validated using bootstrap resampling method. Differential treatment effects between risk categories derived from the nomogram were evaluated among all women enrolled through interaction test between treatment and risk category. A total of 1735 women were included and the final model from 866 women treated by placebo identified the following three factors associate with distant recurrence: tumor size, nodal status, and presence of cardiovascular disease. The nomogram derived from the final model exhibited good discrimination power with a bootstrap-corrected concordance index of 0.71 and, importantly, identified 64% of low risk patients in whom extended treatment has limited benefit. Interaction between treatment and risk category derived from the nomogram was significant (p = 0.04). A nomogram with good performance may be used to accurately predict distant recurrence risk and also benefits with extended treatment after 5 years of aromatase inhibitors. Future independent validation of the proposed nomogram is warranted. NCT00754845

Circulating estrogens in breast cancer patients and survivors are often extremely low due to menopause and estrogen-reducing cancer treatments. Simultaneously, circulating inflammatory markers, and inflammatory proteins in brains of rodent tumor models, can be elevated and correlate with debilitating neurological and psychological comorbidities. Because estrogen has anti-inflammatory properties in the brain, we hypothesized that mammary tumor-induced neuroinflammation is driven, in part, by reduced brain estrogen signaling. An ovariectomized mouse model of postmenopausal breast cancer utilizing the ERα-positive 67NR mammary tumor cell line was used for these experiments. A novel, orally bioavailable, and brain penetrant ERβ agonist was administered daily via oral gavage. Following treatment, estrogen-responsive genes were measured in brain regions. Central and circulating inflammatory markers were measured via RT-qPCR and a multiplex cytokine array, respectively. We present novel findings that peripheral mammary tumors alter estrogen signaling genes including receptors and aromatase in the hypothalamus, hippocampus, and frontal cortex. Mammary tumors induced peripheral and central inflammation, however, pharmacological ERβ activation was not sufficient to reduce this inflammation. Data presented here suggest that compensating for low circulating estrogen with ERβ brain activation is not sufficient to attenuate mammary tumor-induced neuroinflammation, and is therefore not a likely candidate for the treatment of behavioral symptoms in patients. The novel finding that mammary tumors alter estrogen signaling-related genes is a clinically relevant advancement to the understanding of how peripheral tumor biology modulates neurobiology. This is necessary to predict and prevent behavioral comorbidities (e.g., cognitive impairment) prevalent in cancer patients and survivors.

Environmental degradation due to anthropogenic contaminants, particularly the heavy metals are the major concern behind the disappearance of Small Indigenous Fishes (SIFs) from their natural habitats. The present study was aimed to comprehend the underline causes of disappearance of SIFs from their native habitat due to heavy metal by using the environmentally relevant dose of cadmium (Cd) on reproductive potential of zebrafish (Danio rerio) as a model SIF. For this purpose, the effects of Cd exposure on the differential gene expression of aromatase (cyp19b), estrogen receptors (erα, erβa, erβb) and vitellogenin (vtg1) were examined using Reverse Transcriptase Polymerase Chain Reaction (RTPCR) for both male and female fishes; also, the interaction between Cd and estrogen receptor protein were analysed using molecular docking method. The result of the present study showed that, chronic exposure Cd downregulates the expression of Aromatase gene in both male and female fishes by 1.9 and 2.04 folds respectively. The expression of erα was decreased significantly in the female fishes but in case of males the expression was found to be increased and eventually lead to an increased vitellogenin (vtg) expression. Unlike erα, the expression of both erβ isoforms, i.e. erβa and erβb, were found to be significantly upregulated in female fishes and eventually they lead to decreased 1.33 fold vtg production. The in-silico study revealed that Estrogen receptor possess several metal-ion binding sites such as GLU 74, SER 144, SER150, HIS 185, HIS 190, GLU 188, ASP 280, TYR 287 and ASP 318, among them Cd actively interacts with HIS 190, HIS 185 and blocks the estrogen hormone binding site on the receptor protein. In conclusion, our results revealed that Cd as a potent endocrine disruptor can cause long term changes to the reproductive axis of both male and female D. rerio leading to lower reproductive potential.

Estrogenic signaling is an important focus in studies of gonadal and brain sexual differentiation in fishes and vertebrates generally. This study examined variation in estrogenic signaling (1) across three sexual phenotypes (female, female-mimic initial phase [IP] male, and terminal phase [TP] male), (2) during socially-controlled female-to-male sex change, and (3) during tidally-driven spawning cycles in the protogynous bluehead wrasse (Thalassoma bifasciatum). We analyzed relative abundances of messenger RNAs (mRNAs) for the brain form of aromatase (cyp19a1b) and the three nuclear estrogen receptors (ER) (ERα, ERβa, and ERβb) by qPCR. Consistent with previous reports, forebrain/midbrain cyp19a1b was highest in females, significantly lower in TP males, and lowest in IP males. By contrast, ERα and ERβb mRNA abundances were highest in TP males and increased during sex change. ERβa mRNA did not vary significantly. Across the tidally-driven spawning cycle, cyp19a1b abundances were higher in females than TP males. Interestingly, cyp19a1b levels were higher in TP males close (~1 h) to the daily spawning period when sexual and aggressive behaviors rise than males far from spawning (~10-12 h). Together with earlier findings, our results suggest alterations in neural estrogen signaling are key regulators of socially-controlled sex change and sexual phenotype differences. Additionally, these patterns suggest TP male-typical sociosexual behaviors may depend on intermediate rather than low estrogenic signaling. We discuss these results and the possibility that an inverted-U shaped relationship between neural estrogen and male-typical behaviors is more common than presently appreciated.

Nearly 80% of all breast cancers are estrogen receptor positive (ER+) and require the activity of this transcription factor for tumor growth and survival. Thus, endocrine therapies, which target the estrogen signaling axis, have and will continue to be the cornerstone of therapy for patients diagnosed with ER+ disease. Several inhibitors of ER activity exist, including aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders/down-regulators (SERDs), and ER proteolysis-targeting chimeras (ER PROTACs); drugs which differ in the mechanism(s) by which they inhibit this signaling pathway. Notwithstanding their significant impact on the management of this disease, resistance to existing endocrine therapies remains a major impediment to durable clinical responses. Although the mechanisms of resistance are complex and varied, dependence on ER is typically retained after progression on SERMs and AIs, suggesting that ER remains a bona fide therapeutic target. The discovery and development of orally bioavailable drugs that eliminate ER expression (SERDs and ER PROTACs) will likely aid in treating this growing patient population. All of the existing endocrine therapies were developed with the intent of inhibiting the cancer cell intrinsic actions of ER and/or with the objective of achieving extreme estrogen deprivation and most achieve that goal. A longstanding question that remains to be addressed, however, is how actions of existing interventions extrinsic to the cancer cells influence tumor biology. We believe that these issues need to be addressed in the development of strategies to develop the next generation of ER-modulators optimized for positive activities in both cancer cells and other cells within the tumor microenvironment (TME).

We report and review the clinical effectiveness of aromatase inhibitors in a patient with refractory, recurrent and infiltrating endometriosis. We demonstrate excellent clinical, radiological and endoscopic responses after failure of multiple other modalities. Our case and the literature show that single-agent letrozole is capable to treat deep infiltrative endometriosis involving the rectum and the urinary tract. The use of aromatase inhibitor treatment of endometriosis in postmenopausal women makes sense, is safe and is well tolerated. Difficult cases of deep infiltrative endometriosis might require use of combined surgical and medical treatment modalities. Multidisciplinary involvement of the gynecologist, bowel surgeon, urologist and invasive radiologist might be needed. Aromatase inhibitors should be considered to be an integral part of the armamentarium in the management of women with endometriosis, especially in refractory cases that have failed conventional therapeutic modalities.

Duplication of MECP2 (methyl-CpG-binding protein 2) gene causes a serious neurological and developmental disorder called MECP2 duplication syndrome (MDS), which is usually found in males. A previous clinical study reported that MDS patient has precocious puberty with hyperandrogenism, suggesting increased MeCP2 may cause male hyperandrogenism. Here we use an MDS mouse model and confirm that MECP2 duplication significantly upregulates androgen levels. We show for the first time that MeCP2 is highly expressed in the Leydig cells of testis, where androgen is synthesized. Mechanistically, MECP2 duplication increases androgen synthesis and decreases androgen to estrogen conversion through either the upregulation of luteinizing hormone receptor (LHCGR) in testis, as a result of MeCP2 binds to G-quadruplex structure of Lhcgr promoter and recruits the transcription activator CREB1 or the downregulation of the expression of aromatase in testis by binding the CpG island of Rorα, an upstream regulator of aromatase. Taken together, we demonstrate that MeCP2 plays an important role in androgen synthesis, supporting a novel non-CNS function of MeCP2 in the process of sex hormone synthesis.

Topical 17-beta-estradiol (E2) regulates the hair cycle, hair shaft differentiation, and sebum production. Vitamin A also regulates sebum production. Vitamin A metabolism proteins localized to the pilosebaceous unit (PSU; hair follicle and sebaceous gland); and were regulated by E2 in other tissues. This study tests the hypothesis that E2 also regulates vitamin A metabolism in the PSU. First, aromatase and estrogen receptors localized to similar sites as retinoid metabolism proteins during mid-anagen. Next, female and male wax stripped C57BL/6J mice were topically treated with E2, the estrogen receptor antagonist ICI 182,780 (ICI), letrozole, E2 plus letrozole, or vehicle control (acetone) during mid-anagen. E2 or one of its inhibitors regulated most of the vitamin A metabolism genes and proteins examined in a sex-dependent manner. Most components were higher in females and reduced with ICI in females. ICI reductions occurred in the premedulla, sebaceous gland, and epidermis. Reduced E2 also reduced RA receptors in the sebaceous gland and bulge in females. However, reduced E2 increased the number of retinal dehydrogenase 2 positive hair follicle associated dermal dendritic cells in males. These results suggest that estrogen regulates vitamin A metabolism in the skin. Interactions between E2 and vitamin A have implications in acne treatment, hair loss, and skin immunity.

Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor positive (ER+) breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17β-dihydro-EXE (17β-DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione-S-transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17β-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17β-DHE, with glutathionylated EXE and 17β-DHE conjugates detected by ultra-performance liquid chromatography-tandem mass spectrometry. GSTA1, GSTM3, and GSTM1 were active against EXE while only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a KM value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (ClINT=0.14 nl·min-1·mg-1). The KM values observed for human liver cytosol against EXE (46 µM) and 17β-DHE (77 µM) were similar to that observed for recombinant GSTA1 (53 µM and 30 µM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 comprised 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17β-DHE. Significance Statement Most previous studies related to the metabolism of the aromatase inhibitor, exemestane (EXE), have focused mainly on phase I metabolic pathways as well as the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic GST activity against EXE and 17β-DHE and to identify GSTA1 and GSTM1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.

Sex steroid hormones act on hypothalamic kisspeptin neurons to regulate reproductive neural circuits in the brain. Kisspeptin neurons start to express estrogen receptorsin utero, suggesting steroid hormone action on these cells early during development. Whether neurosteroids are locally produced in the embryonic brain and impinge onto kisspeptin/reproductive neural circuitry is not known. To address this question, we analyzed aromatase expression, a key enzyme in estrogen synthesis, in male and female mouse embryos. We identified an aromatase neuronal network comprising ∼6000 neurons in the hypothalamus and amygdala. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male arcuate aromatase neurons convert testosterone to estrogen to regulate kisspeptin neuron activity. We provide spatiotemporal information on aromatase neuronal network development and highlight a novel mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing estrogen receptors.SIGNIFICANCE STATEMENTSex steroid hormones, such as estradiol, are important regulators of neural circuits controlling reproductive physiology in the brain. Embryonic kisspeptin neurons in the hypothalamus express steroid hormone receptors, suggesting hormone action on these cellsin utero. Whether neurosteroids are locally produced in the brain and impinge onto reproductive neural circuitry is insufficiently understood. To address this question, we analyzed aromatase expression, a key enzyme in estradiol synthesis, in mouse embryos and identified a network comprising ∼6000 neurons in the brain. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male aromatase neurons convert testosterone to estradiol to regulate kisspeptin neuron activity.

Most experimental studies on sexual signal regulation via hormone manipulation have focused on male signals, yet female signals demonstrate substantial phenotypic variation and hormone-dependent expression. Female red-sided garter snakes (Thamnophis sirtalis parietalis) produce a skin-based sex pheromone used by males in mate selection. The principle female sex steroid, 17 β-estradiol, controls pheromone production in snakes, but studies manipulating female garter snakes have produced conflicting results, relied on behavioral tests with males in the laboratory, and did not quantify pheromone expression. Because aromatase is the terminal enzyme in estradiol biosynthesis, we hypothesized that female garter snakes rely on aromatase to ultimately control pheromone production during the annual cycle of this species. To test this, we used a known pharmacological inhibitor of aromatase, fadrozole (FAD). Wild-caught female garter snakes were chronically treated via subcutaneous injections of either FAD (1.0 mg kg-1 ) or saline (control) for six months in the laboratory during the active period of the annual cycle then hibernated. In two separate field bioassays the next spring at the den site, FAD females received approximately 50% less courtship from wild, sexually active male garter snakes compared to SHAM females. Pheromone analysis revealed that four of the largest, unsaturated methyl ketones were specifically downregulated in FAD females, indicating that aromatase action is a crucial, permissive step in the maintenance of female attractivity.

Introduction Adrenocortical carcinoma rarely secretes estrogens, and little is known regarding the mechanism of intra-tumor estrogen production. We report an estrogen-secreting adrenocortical carcinoma in a postmenopausal woman, where comprehensive immunohistochemical analyses of the resected tumor revealed disorganized steroidogenesis. Case presentation A 68-year-old woman presented with postmenopausal vaginal bleeding and was found to have a left adrenal tumor. Serum estradiol and testosterone were elevated but they normalized after resection of the tumor, suggestive of adrenocortical carcinoma with disorganized steroidogenesis. Immunohistochemical analyses revealed that the tumor expressed aromatase which converts androgens into estrogens. Furthermore, the tumor lacked 17βHSD2, which converts estradiol to estrone, suggesting that estradiol accumulated as the final product of the tumor's steroidogenic pathway. Conclusion The capability of adrenocortical carcinoma to produce estrogen can be demonstrated by comprehensive immunohistochemical analyses of steroidogenic enzymes, such as those reported here.

The trade-offs between innovation and pharmaceutical access are central to the policy debate on drug pricing. High prices may limit access, result in medication underuse, and negatively affect outcomes. Generic drugs make treatments more affordable. Prior research measured access as utilization without a defined population that should receive certain drugs, it is unknown whether generic entry reduces underuse and thus improves access.To measure changes in access (use, timeliness) with the introduction of three generic aromatase inhibitors (AIs, oral breast cancer drugs) between June 2010 and June 2011.This population-based study included 93,650 older (65+) women diagnosed with hormone receptor-positive breast cancer between 2007 and 2013 in the Surveillance, Epidemiology and End Results-Medicare linked database. We examined changes in access with generic entry for initiation of any adjuvant hormonal therapy drug (AIs or tamoxifen) within one year of diagnosis, time from diagnosis to initiation, and choice of initial therapy.Among 93,650 newly diagnosed breast cancer cases, 67,372 initiated one of the four drugs. With generic entry, initiation rates increased from 69.5% to 74.3%, but non-initiation remained high (up to 25.7%). After controlling for demographics, clinical factors, and insurance coverage, the probability of initiation increased by 4.6 percentage points (P 0.001, 95%CI: [4.1,5.2]) after generic entry. With generic entry, estimated time to initiation decreased by 0.3 months (P 0.001, 95%CI: [0.2,0.3]) from 4.1 months, and the probability of choosing AIs over tamoxifen increased by 5.9 percentage points (P 0.001, 95%CI: [5.3,6.5]). Patterns did not substantially differ by level of cost-sharing.Generic entry of AIs was associated with increased probability of receiving recommended treatments, timeliness of treatment, and the probability of receiving clinically preferred treatments. Price changes with generic entry only partially explained these improvements. High non-initiation rates after generic entry suggest prices are not the sole determinant of access.

Background Sexual differentiation can occur after exposure to aromatase into the left gonad at 6.5 days of incubation. Aromatase inhibitors work by inhibiting the action of the aromatase, which converts androgens into estrogens by a process called aromatization. Objectives The aim of this study was to investigate the effect of in ovo exposure to the aromatase inhibitor from tomato and garlic extract on sexual differentiation and embryonic development in chicken embryos. Methods Three hundred eggs divided into five groups: Control 1 (CO; no injection); control 2 distilled water, DW; 0.1 ml/egg); garlic extract (GAR; 0.1 mg/egg); tomato extract (TOM; 0.1 mg/egg); and garlic and tomato extract mixed (ATM, 0.1 ml/egg). The solution was prepared and injected into the albumin from the thin end of the eggs on day five by using a 1 ml syringe with a 23‐gauge needle. The embryonic test (embryo/egg weight) conducted at 7, 14 and 17 days of incubation. After hatching, feather sexing conducted to determine the initial male. Chicks sex was later confirmed on day 42 by an optical microscope lens. Results The results revealed that there was a significant increase (p, The aim of this study was to investigate the effect of in ovo exposure to the aromatase inhibitor of tomato and garlic extract on sexual differentiation and embryonic development of chicken embryos. Sexual differentiation can occur after exposure to aromatase into the left gonad at 6.5 days of incubation. Aromatase inhibitors work by inhibiting the action of the aromatase, which converts androgens into estrogens by a process called aromatization.

Objective The risks of ovarian hyperstimulation syndrome (OHSS) involve high estrogen (E2) levels. We report two breast cancer patients with polycystic ovarian syndrome who underwent fertility preservation and had severe OHSS; their E2 levels were lowered using aromatase inhibitors (AI). Case Reports A 36-year-old woman underwent controlled ovarian stimulation (COS) with AI and cryopreserved 10 blastocysts. She was hospitalized with OHSS (E2 = 139.1 pg/mL). She improved with infusion alone. A 31-year-old woman underwent COS with AI and cryopreserved 8 blastocysts. She was hospitalized for OHSS (E2: 429 pg/mL). Her vascular endothelial growth factor (VEGF) levels were high (62 pg/mL) at 8 days after the procedure. She needed hospitalization for 9 days. The planned adjuvant therapy was delayed for a week in both cases. Conclusion Elevated VEGF levels should be considered as a risk factor of OHSS even if E2 levels are low with AI treatment.

Background The incidence of bladder cancer (BCa) in male is approximately three to four times higher than in female, but the oncological outcomes in female patients with BCa are significantly worse than in male patients. Although many biomarkers have been identified in recent decades to predict the prognosis of BCa patients, few of them are able to distinguish the prognosis of BCa patients with gender difference. Aromatase encoded by the CYP19A1 gene catalyzes the conversion of androgens to estrogens. In this study, we investigate the prognosis significance of CYP19A1 expression considering the gender difference in BCa patients from four available public databases. Methods Four available public databases of BCa, including GSE13507, TCGA-BLCA, E-MTAB-4321, and E-MTAB-1803, were utilized in this analysis. The overall survival (OS) and progression-free survival (PFS) in different stages and genders were evaluated using the Kaplan-Meier analysis based on the optimal cut-off values of CYP19A1 expression. Then, Gene Set Enrichment Analysis (GSEA) were further performed to explore the potential biologic pathways by altering CYP19A1 expression in BCa patients. Results The results showed that patients with high CYP19A1 expression had a poorer outcome compared with those with low expression in both BCa cohorts in general. Higher CYP19A1 expression in male patients were significantly associated with shorter survival for either non-muscle-invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC). However, female NMIBC patients with high CYP19A1 expression were identified to have a better prognosis, whereas high CYP19A1 expression in female MIBC patients were significantly associated with poorer survival. The result of the GSEA showed that different outcomes in female and male patients with NMIBC were related to the interaction of CYP19A1 and the cell-cycle-related pathways. Conclusions These findings demonstrated that CYP19A1 expression might have a potential role in distinguishing the prognosis of female BCa patients dependent on tumor stage. Our results provide new insights for aromatase-mediated BCa therapy.

Objectives Epicardial adipose tissue (EAT) aromatase converts androstenedione and other adrenal androgens into oestrogens. The locally produced oestradiol (E2) may have cardiovascular protective effects. Little is known about the relationship between EAT aromatase level and coronary heart disease (CHD). Here, we compared EAT aromatase levels in CHD versus non-CHD patients and assessed the relationship between EAT aromatase levels and lesion degree in the coronary arteries. Methods EAT and blood specimens were obtained from patients undergoing thoracotomy prior to cardiopulmonary bypass. Serum E2 levels were obtained from our hospital laboratory. EAT aromatase expression was determined by RT-qPCR and ELISA assays. All patients underwent coronary angiography and the level of coronary lesions was evaluated with the SYNTAX score. Results Compared with non-CHD patients, CHD patients had lower EAT aromatase mRNA and protein levels. In the CHD patients, EAT aromatase and oestrogen levels negatively correlated with the severity of coronary artery disease. Conclusions Our data revealed that reduced EAT aromatase levels correlated with coronary atherosclerotic lesions. Reduced EAT aromatase protein levels may aggravate the severity of atherosclerosis. Future studies should investigate the mechanisms regulating aromatase expression in epicardial fat.

Foxo1, a member of Foxo transcription factor family, is involved in a number of physiological processes including metabolism, cell cycle progression, aging, and apoptosis. In the ovarian granulosa cell of mouse, Foxo1 is implicated to inhibit the expression of Cyp19a1, a gene encoding the aromatase that converts androgens into estrogens. Currently, the information about the expression and physiological relevance of Foxo1 homologues in the ovary of teleosts is scarce. In the present study, cDNAs encoding two forms of Foxo1, Foxo1a and Foxo1b, were isolated from the orange-spotted grouper. Phylogenetic analysis indicated that the orange-spotted groupers Foxo1a and Foxo1b were closely related to the counterparts of the ricefield eel. RT-PCR analysis showed that the orange-spotted groupers foxo1a and foxo1b were expressed in a wide range of tissues, with high levels detected in the brain regions, liver, and intestine. Quantitative real-time PCR analysis showed similar expression profiles for cyp19a1a, foxo1a, and foxo1b in the ovary during development from the primary growth to mature stages, with peak values detected at the vitellogenic stage. In situ hybridization detected mRNA of foxo1a, foxo1b, and cyp19a1a in granulosa cells surrounding vitellogenic oocytes. In vitro transfection showed that both Foxo1a and Foxo1b upregulated the orange-spotted grouper cyp19a1a promoter activities, possibly through the conserved Foxo binding site. Collectively, these results suggest that both Foxo1a and Foxo1b may be involved in the regulation of the ovarian functions in the orange-spotted grouper and the physiological roles of Foxo1 homologues in the ovary may be diversified in vertebrates.

Objectives Aromatase deficiency is a rare autosomal recessive disease that results in the absence of aromatase. In females it presents with ambiguous genitalia and lack of secondary sexual characteristics during puberty. Aromatase deficiency is not attributed to any specific population, but it is more commonly seen in consanguineous parents. Herein, we report the first Old Order Mennonite family with that diagnosis. Case presentation Our proband is an Old Order Mennonite female born with ambiguous genitalia who was identified to carry novel homozygous variant in the CYP19A1 gene c.1304G>A (p. Arg435His). Her older brother was later confirmed with the same genetic diagnosis. Conclusions Recognizing the cultural sensitivity, unrecognized affected cases, and late presentation of males affected with aromatase deficiency, this condition may be more prevalent than believed in that population.

Glioblastoma multiforme (GBM) is the most fatal cancer among brain tumors, and the standard treatment of GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ) chemotherapy. However, tumors always recur due to the developing drug resistance. It has been shown that neurosteroids, including dehydroepiandrosterone and 17β-estradiol, are synthesized in TMZ-resistant GBM tumors. Therefore, we sought to explore the possible role of 17β-estradiol in the development of drug resistance in GBM. Bioinformatics analysis revealed that aromatase/cytochrome P450 19A1 expression was gradually increased in the development from normal, astrocytoma to GBM. The level of 17β-estradiol was significantly increased in TMZ-resistant cells characterized by ultra performance liquid chromatography-tandem mass spectrometry. Furthermore, 17β-estradiol attenuated TMZ-induced cell death and reduced reactive oxygen species production by mitochondria. In addition, 17β-estradiol attenuated oxidative stress by increasing the expression of superoxide dismutase 1/2, catalase, and nuclear factor erythroid 2-related factor (NRF) 2. We found that NRF2 expression was essential for the induction of drug resistance by 17β-estradiol through the reduction of oxidative stress in GBM.

Glomerella fusarioides-mediated structural modifications of steroidal anabolic drug, oxandrolone (1), yielded a new product 17β,11α-dihydroxy-17α-methylandrosta-2-oxa-4-ene-3-one (2). Similarly, Cunninghamella elegans-catalyzed transformation of a steroidal anti-epileptic drug ganaxolone (3) afforded a new metabolite 14α-hydroxy-5α-pregnan-1-ene-3,20-dione (4). Structures of new metabolites 2 and 4 were deduced via 1D-, and 2D-NMR, HREI-, HRFAB-MS, and IR spectroscopic techniques. New derivative 2 showed a remarkable anti-aromatase activity with an IC50 = 0.6 ± 0.005 μM, when compared to substrate 1 (IC50 = 0.808 ± 0.07 μM), and standard anti-cancer drug exemestane (IC50 = 0.232 ± 0.031 μM). Ganaxolone (3) (IC50 = 13.76 ± 2.00 μM) also showed a good activity against human aromatase enzyme, while its derivative 4 was found to be inactive. Inhibition of aromatase enzyme is a key approach in the treatment of estrogen positive breast cancers. Compounds 1-4 were non-cytotoxic to 3T3 cell line (mouse fibroblast).

Obesity is a risk factor for the development of post-menopausal breast cancer. Breast white adipose tissue (WAT) inflammation, which is commonly found in women with excess body fat, is also associated with increased breast cancer risk. Both local and systemic effects are probably important for explaining the link between excess body fat, adipose inflammation and breast cancer. The first goal of this cross-sectional study of 196 women was to carry out transcriptome profiling to define the molecular changes that occur in the breast related to excess body fat and WAT inflammation. A second objective was to determine if commonly measured blood biomarkers of risk and prognosis reflect molecular changes in the breast. Breast WAT inflammation was assessed by immunohistochemistry. Bulk RNA-sequencing was carried out to assess gene expression in non-tumorous breast. Obesity and WAT inflammation were associated with a large number of differentially expressed genes and changes in multiple pathways linked to the development and progression of breast cancer. Altered pathways included inflammatory response, complement, KRAS signaling, tumor necrosis factor α signaling via NFkB, interleukin (IL)6-JAK-STAT3 signaling, epithelial mesenchymal transition, angiogenesis, interferon γ response and transforming growth factor (TGF)-β signaling. Increased expression of several drug targets such as aromatase, TGF-β1, IDO-1 and PD-1 were observed. Levels of various blood biomarkers including high sensitivity C-reactive protein, IL6, leptin, adiponectin, triglycerides, high-density lipoprotein cholesterol and insulin were altered and correlated with molecular changes in the breast. Collectively, this study helps to explain both the link between obesity and breast cancer and the utility of blood biomarkers for determining risk and prognosis.

Background: Aromatase, a cytochrome P450 hemoprotein that is responsible for estrogen biosynthesis by conversion of androgens into estrogens, has been an attractive target in the treatment of hormonedependent breast cancer. Design of new steroidal aromatase inhibitors becomes imperative. Objective: Synthesis and biological evaluation of two classes of structurally and functionally diverse D-ring pregnenolone pyrazoles as type I aromatase inhibitors and antiproliferative agents. Methods: Pregnenolone (1) was converted to 3β-hydroxy-21-hydroxymethylidenepregn-5-en-20-one (2), which upon cyclization with phenylhydrazine generated regioisomeric pairs of pyrazoles 4 and 5. Further, Knoevenagel condensation of pregnenolone (1) with 3-oxo-3-phenylpropanenitrile (6) produced 2-benzoyl-3-(3b-hydroxyandrostan- 5-ene-20-ylidene)-but-2-enenitrile (7), which upon cyclization with hydrazine or phenylhydrazine generated the pyrazoles 8 and 9. All new steroidal derivatives were tested for their aromatase inhibition activity using Dibenzylfluorescein (DBF) based fluorescence assay developed by Stresser et al. Antiproliferative activities were measured using Sulforhodamine B assay. The activities were promising and there was a coherence between aromatase inhibitory and antiproliferative activities. Results: The study reveals the immense potential of pregnenolone pyrazoles as aromatase inhibitors for the treatment of breast cancer. Molecular docking studies proved efficient binding of the new steroidal analogs on human placental aromatase. Conclusions: n the overall study, most of the compounds exhibited potential activity for the treatment of hormone dependent breast cancer. Compounds 4c and 4d were found to be the most promising pharmacons. Furthermore, compounds 4c and 4d were applied for their molecular docking study on human placental aromatase to predict their possible binding modes with the enzyme. These studies revealed that such molecules have high scope and potential for further investigation towards the treatment of estrogen dependent breast cancer.

Inhibition of estrogen synthesis is an integral component of the frontline pharmacologic therapy for the treatment of estrogen receptor positive cancers. However, there is currently no direct, high-throughput-ready assay for aromatase (also known as CYP19A1) that can be performed in live cells. Herein we present a cell-based assay that allows for multiplexed assessment of enzyme activity, protein half-life, cell viability, and identification of inhibitors with slow off-rates.

Diffuse optical tomography breast imaging system (DOTBIS) non-invasively measures tissue concentration of hemoglobin, which is a potential biomarker of short-term response to neoadjuvant chemotherapy. We evaluated whether DOTBIS-derived measurements are modifiable with targeted therapies, including AKT inhibition and endocrine therapy. We conducted a proof of principle study in seven postmenopausal women with stage I-III breast cancer who were enrolled in pre-surgical studies of the AKT inhibitor MK-2206 (n = 4) or the aromatase inhibitors exemestane (n = 2) and letrozole (n = 1). We performed DOTBIS at baseline (before initiation of therapy) and post-therapy in the affected breast (tumor volume) and contralateral, unaffected breast, and measured tissue concentrations (in μM) of total hemoglobin (ctTHb), oxyhemoglobin (ctO2Hb), and deoxyhemoglobin (ctHHb), as well as water fraction (%). We found consistent decreases in DOTBIS-measured hemoglobin concentrations in tumor volume, with median percent changes for ctTHb, ctHHb, ctO2Hb, and water fraction for the entire cohort of − 27.1% (interquartile range [IQR] 37.5%), − 49.8% (IQR 29.3%), − 33.5% (IQR 47.4%), and − 3.6% (IQR 10.6%), respectively. In the contralateral breast, median percent changes for ctTHb, ctHHb, ctO2Hb, and water fraction were + 1.8% (IQR 26.7%), − 8.6% (IQR 29.3%), + 6.2% (IQR 29.5%), and + 1.9% (IQR 30.7%), respectively. We demonstrated that DOTBIS-derived measurements are modifiable with pre-surgical AKT inhibition and endocrine therapy, supporting further investigation of DOTBIS as a potential imaging assessment of response to neoadjuvant targeted therapies in early stage breast cancer.

Introduction Aromatase is a CYP450 enzyme that catalyses the conversion of androgens into oestrogens, where the decrease in the production of oestrogens aided by aromatase inhibitors is considered a target in post-menopausal breast cancer therapy. TLC-bioautography is a technique employed for combining chromatographic separations on TLC plates with bioassays. This is the first report to evaluate aromatase inhibitory activity using this technique. Objectives The aim of this study is to develop and validate a new TLC-bioautographic method for determination of aromatase inhibitory activity in 14 plant extracts. Two quantitation methods, the peak area method and reciprocal iso-inhibition volume (RIV) method, were compared and investigated to attain reliable results. Factors affecting the enzymatic reaction (temperature, pH, enzyme and substrate concentrations … etc.) were also investigated to attain the optimum parameters. Methodology TLC assisted by digital image processing was implemented for quantitative estimation of the aromatase inhibition of 14 plant extracts using chrysin as positive control. The fluorometric substrate dibenzyl fluorescein (DBF) was utilised for the assay, where inhibitory compounds were visualised as dark spots against a blue fluorescent background. Two software programs, Sorbfil® videodensitometer (in the peak area method) and ImageJ® (in the RIV method), were thoroughly validated using the International Council on Harmonisation (ICH) guideline and used for quantitation. Results The RIV method showed superiority over the peak area method in the quantitation results of the tracks with non-homogenous background with %RSD values of 0.98 and 1.49 compared with 2.86 and 3.58, respectively. Further, the methods allow the comparison of the activity of different unknown inhibitory compounds without the need for a reference or a positive control. Conclusion Using the TLC-bioautographic method by image processing combined with the RIV quantitation method, simultaneous separation and quantitation of aromatase inhibitory components could be applied to estimate the relative activity of various plant extracts.

The cryopreservation of secondary follicles (SF) is a promising alternative to preserve the reproductive potential both in humans and animals in situations in which the transplantation of ovarian tissue is not possible. The objective of the present study was cryopreserved SF isolated sheep. Beyond follicular morphology, viability and development, we investigated proteins related to steroidogenic function and basement membrane remodeling [metalloproteinases 2 (MMP-2) and 9 (MMP-9)] in fresh SF (FSF) and vitrified SF (VSF) followed by in vitro culture for 6 (D6) or 12 days (D12). The percentage of intact follicles, follicular and oocyte diameter of the VSF were lower than FSF on both days of culture (P

Aromatase plays a significant role in the progression of estrogen receptor-positive (ER-positive) breast cancer. The adverse side effects of currently used aromatase inhibitors (AIs) necessitate the development of new AIs that are more active, selective, and less toxic. This study used a computational approach to screen 503 natural compounds ZINC database against the aromatase active site. The best scoring hits ZINC69482055, ZINC69482510, and ZINC406719 exhibited strong binding with aromatase, with binding energy values of -8.45, -10.35, and -8.75 kcal/mol, respectively, which is comparatively higher than that of the control compound Anastrozole (-6.43 kcal/mol). Docking analysis showed that the selected hits interacted with the crucial residues of the aromatase active site. This study suggested that these compounds could be used as possible AIs in the cure of breast cancer. Hands-on bench work validation is needed to optimize these compounds as AIs.

In this study, we aimed to clone brain-derived Cyp19b and ovary-derived Cyp19a the P450 aromatase gene isoforms and to indicate the expression levels of these genes in the hypothalamus and ovary tissues from reproductively arrested ovarian development (RA) and non-arrested ovarian development (RN) Alburnus tarichi from Lake Van, Turkey. The expression levels of Cyp19b and Cyp19a genes were predominant in the hypothalamus and ovary, respectively. The level of Cyp19b mRNA was significantly lower in the hypothalamus and ovary from RA fish than in the hypothalamus and ovary from RN fish (P, Bu çalışmanın amacı, Van Gölü’nden yakalanan dişi Alburnus tarichi’de P450 aromataz izoformları olan beyin kaynaklı Cyp19b ve ovaryum kaynaklı Cyp19a genlerini klonlanmak ve üreme bakımından ovaryum gelişimi engellenmiş (RA) ve ovaryum gelişimi normal olan (RN) balıklardan alınan hypothalamus ve ovaryum örneklerinde bu genlerin ekspresyon seviyelerini belirlemektir. Cyp19b gen ekspersyonu beyinde, Cyp19a gen ekspresyonu ise ovaryumda daha fazlaydı. Cyp19b mRNA seviyesi, RA balıkların ovaryum ve hipotalamusunda, RN balıkların ovaryum ve hipotalamusundan belirgin olarak düşüktü (P

Adjuvant treatment for post-menopausal women with early breast cancer (BC) includes aromatase inhibitors (AI), known to decrease bone mineral density (BMD). In this study, we investigate whether denosumab is a valid second option for patients unable to receive standard adjuvant i.v. zoledronic acid (ZA). In total, 212 patients have been evaluated after they did not receive ZA. Of those 194 were included. After evaluation by an endocrinologist, all patients were offered ZA as their first choice and 15% accepted (

OBJECTIVES: To evaluate the ocular surface, meibomian glands and corneal structural changes using in vivo confocal microscopy (IVCM) in patients receiving aromatase inhibitor (AI) therapy due to the breast cancer. METHODS: This prospective observational study included 13 patients undergoing AI therapy. The patients were evaluated before the treatment, at 3- and 6-month timepoints of AI therapy. To examine the ocular surface and tear film, corneal sensitivity (CS) measurement with Cochet–Bonnet Aesthesiometer, tear film break-up time (TBUT), lissamine green (LG) staining, Schirmer I test with anaesthesia (ST) and the ocular-surface disease index (OSDI) questionnaire were performed consecutively. Corneal cell densities and sub-basal nerve plexus were evaluated with IVCM (ConfoScan 4, Nidek, Japan). Finally, quantitative MG drop-out assessment was made using infrared meibography. Shapiro Wilk, Friedman’s and Post-hoc Dunn tests were used for the statistical analysis. RESULTS: TBUT, ST scores, basal epithelium, anterior and posterior keratocytes and endothelial cell densities, long and total sub-basal nerve densities were found to be decreased (p

Investigation of bioactive compounds from the rhizomes of Kaempferia elegans led to the isolation and characterization of ten new diterpenoids, namely, five 12,13-seco-diterpenoids named elegansins A-E (1-5) and five new abietanes, elegansols A-E (6-10), together with seven known diterpenoids (11-17). The structure elucidation of the new compounds was achieved by HRESIMS, NMR, and ECD spectroscopic analysis. Compounds (1-5) are the first examples of 12,13-seco-diterpenoid-type compounds representing a decalin fused dihydropyran skeleton. Plausible biosynthetic pathways for compounds 1-5 are proposed. Aromatase inhibitory activities of all compounds were evaluated, and abieta-8,11,13-trien-11-ol (16) was found to be the most potent aromatase inhibitor with an IC50 value of 3.7 μM.

PURPOSE: Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance. PATIENTS AND METHODS: In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness. RESULTS: High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies. CONCLUSION: Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.