Thalassosterol, a New Cytotoxic Aromatase Inhibitor Ergosterol Derivative from the Red Sea Seagrass Thalassodendron ciliatum.

Thalassodendron ciliatum (Forssk.) Den Hartog is a seagrass belonging to the plant family Cymodoceaceae with ubiquitous phytoconstituents and important pharmacological potential, including antioxidant, antiviral, and cytotoxic activities. In this work, a new ergosterol derivative named thalassosterol (1) was isolated from the methanolic extract of T. ciliatum growing in the Red Sea, along with two known first-reported sterols, namely ergosterol (2) and stigmasterol (3), using different chromatographic techniques. The structure of the new compound was established based on 1D and 2D NMR spectroscopy and high-resolution mass spectrometry (HR-MS) and by comparison with the literature data. The new ergosterol derivative showed significant in vitro antiproliferative potential against the human cervical cancer cell line (HeLa) and human breast cancer (MCF-7) cell lines, with IC50 values of 8.12 and 14.24 µM, respectively. In addition, docking studies on the new sterol 1 explained the possible binding interactions with an aromatase enzyme; this inhibition is beneficial in both cervical and breast cancer therapy. A metabolic analysis of the crude extract of T. ciliatum using liquid chromatography combined with high-resolution electrospray ionization mass spectrometry (LC-ESI-HR-MS) revealed the presence of an array of phenolic compounds, sterols and ceramides, as well as di- and triglycerides.

Boronic-targeted albumin-shell oily-core nanocapsules for synergistic aromatase inhibitor/herbal breast cancer therapy.

Multi-modality strategies of albumin-mediated drug accumulation in tumor, boronate-based active tumor targeting and synergistic cancer therapy were combined together for effective treatment of breast cancer. Herein we report the development of albumin-shell oily-core nanocapsules (NCs), loaded with novel combination of hydrophobic drugs, exemestane (EXE) and hesperetin (HES), for targeted breast cancer therapy. This protein-lipid nanohybrid carrier was successfully fabricated using a simple protein-coating method based on the electrostatic adsorption of negatively charged albumin shell onto the oily core containing cationic surfactant. While EXE was directly encapsulated into the oily core, HES was pre-formulated in the form of phospholipid complex before solubilization in oily phase. In addition to albumin-mediated binding to albondin and SPARC, phenylboronic acid was chemically coupled to the albumin shell to confer additional tumor targeting. The targeted nanocarrier (TNC) demonstrated enhanced internalization into MCF-7 breast cancer cells resulting in synergistic cytotoxic activity with a combination index (CI) of 0.662 and dose reduction index (DRI) of 8.22 and 1.84 for EXE and HES, respectively. In vivo, TNC displayed superior anti-cancer activity in tumor-bearing mice compared to their non-targeted counterparts and the free drug combination. A significant reduction of both tumor volume (7-folds) and Ki67 expression (3-folds) was obtained by the targeted nanocarriers compared to positive control. Overall, the boronic-targeted albumin NCs offer a promising platform for hydrophobic drug combination against cancer therapy.

Aromatase (CYP19) inhibition by biflavonoids obtained from the branches of Garcinia gardneriana (Clusiaceae).

Overexpression of aromatase in breast cancer cells may substantially influence its progression and maintenance. In this sense, the inhibition of aromatase is a key target for the treatment of breast cancer in postmenopausal women. Although several flavonoids had already demonstrated the capacity of inhibiting aromatase activity, the role of biflavonoids as aromatase inhibitors is poorly studied. In this work, the biflavonoids isolated from Garcinia gardneriana, morelloflavone (1), Gb-2a (2) and Gb-2a-7-O-glucose (3) were submitted to in vitro assay to evaluate the aromatase modulatory effect. As results, it was demonstrated that all biflavonoids were able to inhibit the enzyme, with IC50 values ranging from 1.35 to 7.67 µM. This demonstrates that biflavonoids are an important source of scaffolds for the development of new aromatase inhibitors, focusing on the development of new anticancer agents.

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques.

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large-scale production of functional food and nutraceutical ingredients.

Plant derived anti-cancerous secondary metabolites as multipronged inhibitor of COX, Topo, and aromatase: molecular modeling and dynamics simulation analyses.

In the present study, 300 plant derived secondary metabolites (100 each of alkaloid, flavonoid, and terpenoid), have been screened for their anti-cancerous activity through inhibition of selected key enzymatic targets, namely cyclooxygenases (COXs), topoisomerases (Topos), and aromatase by molecular docking approach. Furthermore, the stability of the complexes of top hits, from each class of secondary metabolites, with their respective enzymatic targets was analyzed using molecular dynamics (MD) simulation analyses and binding free energy calculations. Analysis of the results of the docking in light of the pharmacokinetically screened 18 alkaloids, 26 flavonoids, and 9 terpenoids, revealed that the flavonoid, curcumin, was the most potent inhibitor for all the selected enzymatic targets. The stability of the complexes of COX-1, COX-2, Topo I, Topo IIß and aromatase with the most potent inhibitor curcumin and those of the respective drugs, namely ibuprofen, aspirin, topotecan, etoposide, and exemestane were also analyzed through MD simulation analyses which revealed better stability of curcumin complexes than those of respective drugs. Binding energy calculations of the complexes of the curcumin with all the targets, except those of Topos, exhibited lower binding energies for the curcumin complexes than those of respective drugs which corroborated with the results of molecular docking analyses. Thus, the present study affirms the versatile and multipronged nature of curcumin, the traditionally used herbal medicine, as anti-cancer molecule directed against these enzymatic targets.

Nelumal A, the active principle of Ligularia nelumbifolia, is a novel aromatase inhibitor.

Nelumal A, the active principle of Ligularia nelumbifolia was preliminarily tested as an aromatase inhibitors in HEK293 cells transfected with aromatase cDNA and using anastrazole as the reference drug. This screening revealed that it showed an appreciable level of inhibition. Subsequent experiments aimed to evaluate the aromatase activity and expression in KGN cells confirmed that the title natural product, after an incubation of 48 h, compared favourably with anastrazole (1 microM) in the concentration range 10-30 microM. Moreover, nelumal A (30 microM) abolished the aromatase mRNA expression in the same cell line.

Anti-tumor effect of Shu-Gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer cell line ZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo. MATERIALS AND METHODS: The anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay. RESULTS: SGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC50 value of 3.40 mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E1S). Oral administration of 6 g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo. CONCLUSION: Our results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.

Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component.

Rhus verniciflua Stokes has been used as a traditional herbal medicine in Asia. In this study, the effect of R. verniciflua extract on human aromatase (cytochrome P450 19, CYP19) activity was investigated to elucidate the mechanism for the effect of R. verniciflua extract on androgen hormone levels. Androstenedione was used as a substrate and incubated with R. verniciflua extract in cDNA-expressed CYP19 supersomes in the presence of NADPH, and estrone formation was measured using liquid chromatography-tandem mass spectrometry. R. verniciflua extract was assessed at concentrations of 10-1000 µg/mL. The resulting data showed that R. verniciflua extract inhibited CYP19-mediated estrone formation in a concentration-dependent manner with an IC50 value of 136 µg/mL. Subsequently, polyphenolic compounds from R. verniciflua extract were tested to identify the ingredients responsible for the aromatase inhibitory effects by R. verniciflua extract. As a result, butin showed aromatase inhibitory effect in a concentration-dependent manner with an IC50 value of 9.6 µM, whereas the inhibition by other compounds was negligible. These results suggest that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity and butin is a major ingredient responsible for this activity.

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary.

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in-capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced ß-nicotinamide adenine dinucleotide 2'-phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced ß-nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver-Burk equation, the Michaelis-Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.

Immunoprecipitation coupled with HPLC-MS/MS to discover the aromatase ligands from Glycyrrhiza uralensis.

Affinity chromatography, applied to discover the enzyme inhibitors, needs special column with target protein and its carrier. Selection of stationary phase and mobile phase needs careful considerations due to the characteristics of proteins. In this study, a method immunoprecipitation (IP) coupled with HPLC-DAD-MS was developed to discover the aromatase ligands from Glycyrrhiza uralensis. An SB-C18 column was employed to separate target compounds without special consideration in mobile phase. Twenty-one compounds, including isolated compounds 4, 7, 8, 10, 11, 13, 15, 18-20, 23 and non-isolated compounds A-J, were found to have good affinity to aromatase by LC-MS. Seven of them (7, 15, 18, 19, 23, D, E) were detected to bind with aromatase in MCF-7 cells by IP coupled with HPLC-MS/MS. Bioassays disclosed aromatase inhibitory activities of the isolated compounds mentioned above, verifying the efficiency of IP coupled with HPLC-MS/MS as a method to screen aromatase ligands.

New ruthenium(II)-letrozole complexes as anticancer therapeutics.

Novel ruthenium-letrozole complexes have been prepared, and cell viability of two human cancer cell types (breast and glioblastoma) was determined. Some ruthenium compounds are known for their cytotoxicity to cancer cells, whereas letrozole is an aromatase inhibitor administered after surgery to post-menopausal women with hormonally responsive breast cancer. A significant in vitro activity was established for complex 5·Let against breast cancer MCF-7 cells and significantly lower activity against glioblastoma U251N cells. The activity of 5·Let was even higher than that of 4, a compound analogous to the well-known drug RAPTA-C. Results from the combination of 5·Let (or 4) with 3-methyladenine (3-MA) or with curcumin, respectively, revealed that the resultant cancer cell death likely involves 5·Let-induced autophagy.

Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 µM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer.

Depsidones, aromatase inhibitors and radical scavenging agents from the marine-derived fungus Aspergillus unguis CRI282-03.

Three new depsidones ( 1, 3, and 4), a new diaryl ether ( 5), and a new natural pyrone ( 9) (synthetically known), together with three known depsidones, nidulin ( 6), nornidulin ( 7), and 2-chlorounguinol ( 8), were isolated from the marine-derived fungus ASPERGILLUS UNGUIS CRI282-03. Aspergillusidone C ( 4) showed the most potent aromatase inhibitory activity with the IC (50) value of 0.74 µM, while depsidones 1, 3, 6- 8 inhibited aromatase with IC (50) values of 1.2-11.2 µM. It was found that the structural feature of depsidones, not their corresponding diaryl ether derivatives (e.g. 5), was important for aromatase inhibitory activity. Aspergillusidones A ( 1) and B ( 3) showed radical scavenging activity in the XXO assay with IC (50) values of 16.0 and < 15.6 µM, respectively. Compounds 1 and 3- 7 were mostly inactive or showed only weak cytotoxic activity against HuCCA-1, HepG2, A549, and MOLT-3 cancer cell lines.

Synthesis and screening of aromatase inhibitory activity of substituted C19 steroidal 17-oxime analogs.

The synthesis and aromatase inhibitory activity of androst-4-en-, androst-5-en-, 1ß,2ß-epoxy- and/or androsta-4,6-dien-, 4ß,5ß-epoxyandrostane-, and 4-substituted androst-4-en-17-oxime derivatives are described. Inhibition activity of synthesized compounds was assessed using aromatase enzyme and [1ß-3H]androstenedione as substrate. Most of the compounds displayed similar to or more aromatase inhibitory activity than formestane (74.2%). 4-Chloro-3ß-hydroxy-4-androsten-17-one oxime (14, 93.8%) showed the highest activity, while 4-azido-3ß-hydroxy-4-androsten-17-one oxime (17, 32.8%) showed the lowest inhibitory activity for aromatase.

Aromatase inhibitors and antiepileptic drugs: a computational systems biology analysis.

BACKGROUND: The present study compares antiepileptic drugs and aromatase (CYP19) inhibitors for chemical and structural similarity. Human aromatase is well known as an important pharmacological target in anti-breast cancer therapy, but recent research demonstrates its role in epileptic seizures, as well. The current antiepileptic treatment methods cause severe side effects that endanger patient health and often preclude continued use. As a result, less toxic and more tolerable antiepileptic drugs (AEDs) are needed, especially since every individual responds differently to given treatment options. METHODS: Through a pharmacophore search, this study shows that a model previously designed to search for new classes of aromatase inhibitors is able to identify antiepileptic drugs from the set of drugs approved by the Food and Drug Administration. Chemical and structural similarity analyses were performed using five potent AIs, and these studies returned a set of AEDs that the model identifies as hits. RESULTS: The pharmacophore model returned 73% (19 out of 26) of the drugs used specifically to treat epilepsy and approximately 82% (51 out of 62) of the compounds with anticonvulsant properties. Therefore, this study supports the possibility of identifying AEDs with a pharmacophore model that had originally been designed to identify new classes of aromatase inhibitors. Potential candidates for anticonvulsant therapy identified in this manner are also reported. Additionally, the chemical and structural similarity between antiepileptic compounds and aromatase inhibitors is proved using similarity analyses. CONCLUSIONS: This study demonstrates that a pharmacophore search using a model based on aromatase inhibition and the enzyme's structural features can be used to screen for new candidates for antiepileptic therapy. In fact, potent aromatase inhibitors and current antiepileptic compounds display significant - over 70% - chemical and structural similarity, and the similarity analyses performed propose a number of antiepileptic compounds with high potential for aromatase inhibition.

Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors.

Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.

Natural compounds with aromatase inhibitory activity: an update.

Several synthetic aromatase inhibitors are currently in clinical use for the treatment of postmenopausal women with hormone-receptor positive breast cancer. However, these treatments may lead to untoward side effects and so the search for new aromatase inhibitors continues, especially those for which the activity is promoter-specific, targeting the breast-specific promoters I.3 and II. Recently, numerous natural compounds have been found to inhibit aromatase in noncellular, cellular, and IN VIVO studies. These investigations, covering the last two years, as well as additional studies that have focused on the evaluation of natural compounds as promoter-specific aromatase inhibitors or as aromatase inducers, are described in this review.

Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool.

Cytochrome P450 aromatase is a key steroidogenic enzyme that converts androgens to estrogens in vertebrates. There is much interest in aromatase inhibitors (AIs) both because of their use as pharmaceuticals in the treatment of estrogen-sensitive breast cancers, and because a number of environmental contaminants can act as AIs, thereby disrupting endocrine function in humans and wildlife through suppression of circulating estrogen levels. The goal of the current work was to develop a mechanism-based structure-activity relationship (SAR) categorization framework highlighting the most important chemical structural features responsible for inhibition of aromatase activity. Two main interaction mechanisms were discerned: steroidal and non-steroidal. The steroid scaffold is most prominent when the structure of the target chemical is similar to the natural substrates of aromatase - androstenedione and testosterone. Chemicals acting by non-steroidal mechanism(s) possess a heteroatom (N, O, S) able to coordinate the heme iron of the cytochrome P450, and thus interfere with steroid hydroxylation. The specific structural boundaries controlling AI for both analyzed mechanisms were defined, and a software tool was developed that allowed a decision tree (profile) to be built discriminating AIs by mechanism and potency. An input chemical follows a profiling path and the structure is examined at each step to decide whether it conforms with the structural boundaries implemented in the decision tree node. Such a system would aid drug discovery efforts, as well as provide a screening tool to detect environmental contaminants that could act as AIs.

Aromatase inhibitory fatty acid derivatives from the pollen of Brassica campestris L. var. oleifera DC.

Two new fatty acid derivatives, 9Z,12Z,15Z-octadecatrienoic acid sorbitol ester (1) and (10,11,12)-trihydroxy-(7Z,14Z)-heptadecadienoic acid (2), were isolated from the pollen of Brassica campestris L. var. oleifera DC., along with the four known fatty acid derivatives, 9Z,12Z,15Z-octadecatrienamide, N-(2-hydroxyethyl) (3), hexadecanoic acid sorbitol ester (4), 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (5), and 9Z,12Z,15Z-octadecatrienoic acid 2,3-dihydroxypropyl ester (6). Their structures were elucidated by extensive spectroscopic analysis, including 1D- and 2D-NMR as well as HR-ESI-MS experiments. All compounds were tested using a noncellular aromatase assay, and the results showed that some compounds possessed strong inhibitory activity.

Fast three dimensional pharmacophore virtual screening of new potent non-steroid aromatase inhibitors.

Suppression of estrogen biosynthesis by aromatase inhibition is an effective approach for the treatment of hormone sensitive breast cancer. Third generation non-steroid aromatase inhibitors have shown important benefits in recent clinical trials with postmenopausal women. In this study we have developed a new ligand-based strategy combining important pharmacophoric and structural features according to the postulated aromatase binding mode, useful for the virtual screening of new potent non-steroid inhibitors. A small subset of promising drug candidates was identified from the large NCI database, and their antiaromatase activity was assessed on an in vitro biochemical assay with aromatase extracted from human term placenta. New potent aromatase inhibitors were discovered to be active in the low nanomolar range, and a common binding mode was proposed. These results confirm the potential of our methodology for a fast in silico high-throughput screening of potent non-steroid aromatase inhibitors.