In silico identification of novel inhibitors targeting the DNA-binding domain of the human estrogen receptor alpha.

The human estrogen receptor alpha (ERα) is an important regulator in breast cancer development and progression. The frequent ERα mutations in the ligand-binding domain (LBD) can increase the resistance of antiestrogen drugs, highlighting the need to develop new drugs to target ERα-positive breast cancer. In this study, we combined molecular docking, molecular dynamics simulations and binding free energy calculations to develop a structure-based virtual screening workflow to identify hit compounds capable of interfering with the recognition of ERα by the specific response element of DNA. A druggable pocket on the DNA binding domain (DBD) of ERα was identified as the potential binding site. The hits binding modes were further analyzed to reveal the structural characteristics of the DBD-inhibitor complexes. The core structure of the lead molecules was synthesized and was found to inhibit the E2-induced cell proliferation in MCF-7 cell lines. These findings provide an insight into the structural basis of ligand-ERα for alternate sites beyond the LBD-based pocket. The core structure proposed in this study could potentially be used as the lead molecule for further rational optimization of the antiestrogen drug structure with stronger binding of DBD and higher activity.

Dietary bioactive diindolylmethane enhances the therapeutic efficacy of centchroman in breast cancer cells by regulating ABCB1/P-gp efflux transporter.

Overexpression of drug efflux transporters is commonly associated with multidrug-resistance in cancer therapy. Here for the first time, we investigated the ability of diindolylmethane (DIM), a dietary bioactive rich in cruciferous vegetables, in enhancing the efficacy of Centchroman (CC) by modulating the drug efflux transporters in human breast cancer cells. CC is a selective estrogen receptor modulator, having promising therapeutic efficacy against breast cancer. The combination of DIM and CC synergistically inhibited cell proliferation and induced apoptosis in breast cancer cells. This novel combination has also hindered the stemness of human breast cancer cells. Molecular docking analysis revealed that DIM had shown a strong binding affinity with the substrate-binding sites of ABCB1 (P-gp) and ABCC1 (MRP1) drug-efflux transporters. DIM has increased the intracellular accumulation of Hoechst and Calcein, the substrates of P-gp and MRP1, respectively, in breast cancer cells. Further, DIM stimulates P-gp ATPase activity, which indicates that DIM binds at the substrate-binding domain of P-gp, and thereby inhibits its efflux activity. Intriguingly, DIM enhanced the intracellular concentration of CC by inhibiting the P-gp and MRP1 expression as well as activity. The intracellular retaining of CC has increased its efficacy against breast cancer. Overall, DIM, a dietary bioactive, enhances the anticancer efficiency of CC through modulation of drug efflux ABC-transporters in breast cancer cells. Therefore, DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy against human breast cancer.

Bispecific Estrogen Receptor α Degraders Incorporating Novel Binders Identified Using DNA-Encoded Chemical Library Screening.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

ERα-agonist and ERß-antagonist bifunctional next-generation bisphenols with no halogens: BPAP, BPB, and BPZ.

As demonstrated for bisphenol AF (BPAF), the electrostatic halogen bond based on the London dispersion force of halogen atoms was found to be a major driving force of their bifunctional ERα-agonist and ERß-antagonist activities. Because similar electronic effects are anticipated for hydrocarbon groups (alkyl or aryl groups), we hypothesized that bisphenol compounds consisting of such groups also work bifunctionally. In the present study, we examined bisphenol AP (BPAP), B (BPB), and Z (BPZ). After recognizing their considerably strong receptor binding affinities, we evaluated the abilities of BPAP, BPB, and BPZ to activate ERα and ERß in a luciferase reporter gene assay. These bisphenols were fully active for ERα but completely inactive for ERß. When we examined their inhibitory activities for 17ß-estradiol in ERß by two different qualitative and quantitative analytical methods, we found that those bisphenols worked as definite antagonists. Consequently, they were established as bifunctional ERα-agonists and ERß-antagonists. The present structure-activity analyses revealed that the dispersion force works not only on the halogens but also on the hydrocarbon groups, and that it is a major driving force of bifunctional ERα-agonist and ERß-antagonist activities.

Paclitaxel and naringenin-loaded solid lipid nanoparticles surface modified with cyclic peptides with improved tumor targeting ability in glioblastoma multiforme.

The present work describes the systematic development of paclitaxel and naringenin-loaded solid lipid nanoparticles (SLNs) for the treatment of glioblastoma multiforme (GBM). So far only temozolomide therapy is available for the GBM treatment, which fails by large amount due to poor brain permeability of the drug and recurrent metastasis of the tumor. Thus, we investigated the drug combination containing paclitaxel and naringenin for the treatment of GBM, as these drugs have individually demonstrated significant potential for the management of a wide variety of carcinoma. A systematic product development approach was adopted where risk assessment was performed for evaluating the impact of various formulation and process parameters on the quality attributes of the SLNs. I-optimal response surface design was employed for optimization of the dual drug-loaded SLNs prepared by micro-emulsification method, where Percirol ATO5 and Dynasan 114 were used as the solid lipid and surfactant, while Lutrol F188 was used as the stabilizer. Drug loaded-SLNs were subjected to detailed in vitro and in vivo characterization studies. Cyclic RGD peptide sequence (Arg-Gly-Asp) was added to the formulation to obtain the surface modified SLNs which were also evaluated for the particle size and surface charge. The optimized drug-loaded SLNs exhibited particle size and surface charge of 129 nm and 23 mV, drug entrapment efficiency >80% and drug loading efficiency >7%. In vitro drug release study carried out by micro dialysis bag method indicated more than 70% drug was release observed within 8 h time period. In vivo pharmacokinetic evaluation showed significant improvement (p < 0.05) in drug absorption parameters (Cmax and AUC) from the optimized SLNs over the free drug suspension. Cytotoxicity evaluation on U87MG glioma cells indicated SLNs with higher cytotoxicity as compared to that of the free drug suspension (p < 0.05). Evaluation of uptake by florescence measurement indicated superior uptake of SLNs tagged with dye over the plain dye solution. Overall, the dual drug-loaded SLNs showed better chemoprotective effect over the plain drug solution, thus construed superior anticancer activity of the developed nanoformulation in the management of glioblastoma multiforme.

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

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

Binding Assays Using a Benzofurazan-Labeled Fluorescent Probe for Estrogen Receptor-Ligand Interactions.

Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (Kd) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles.

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition.

3-ß-hydroxysteroid-Δ8, Δ7-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.

Determination and analysis of agonist and antagonist potential of naturally occurring flavonoids for estrogen receptor (ERα) by various parameters and molecular modelling approach.

Most estrogen receptor α (ERα) ligands target the ligand binding domain (LBD). Agonist 17ß-estradiol (E2) and tamoxifen (TM, known SERM), bind to the same site within the LBD. However, structures of ligand-bound complexes show that E2 and TM induce different conformations of helix 12 (H12). During the molecular modelling studies of some naturally occurring flavonoids such as quercetin, luteolin, myricetin, kaempferol, naringin, hesperidin, galangin, baicalein and epicatechin with human ERα (3ERT and 1GWR), we observed that most of the ligands bound to the active site pocket of both 3ERT and 1GWR. The docking scores, interaction analyses, and conformation of H12 provided the data to support for the estrogenic or antiestrogenic potential of these flavonoids to a limited degree. Explicit molecular dynamics for 50 ns was performed to identify the stability and compatibility pattern of protein-ligand complex and RMSD were obtained. Baicalein, epicatechin, and kaempferol with 1GWR complex showed similar RMSD trend with minor deviations in the protein backbone RMSD against 1GWR-E2 complex that provided clear indications that ligands were stable throughout the explicit molecular simulations in the protein and outcome of naringin-3ERT complex had an upward trend but stable throughout the simulations and all molecular dynamics showed stability with less than overall 1 Å deviation throughout the simulations. To examine their estrogenic or antiestrogenic potential, we studied the effect of the flavonoids on viability, progesterone receptor expression and 3xERE/3XERRE-driven reporter gene expression in ERα positive and estrogen responsive MCF-7 breast cancer cells. Epicatechin, myricetin, and kaempferol showed estrogenic potential at 5 µM concentration.

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton.

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.

Intimate estrogen receptor-α/ligand relationships signal biological activity.

How does estrogen receptor-α bind its natural ligands - estrogens? How can other molecules mimic estrogens and elicit different estrogenic responses? The answers lie in a complex and intimate chemical biology between ligands and receptor. This delicate interaction at the ligand binding cleft signals, via conformational change, exposure of a specific new charge topography at a second site (Activation Function-2). This, in turn, attracts a regulatory protein which modulates gene expression and controls biological activity.

3,20-Dihydroxy-13α-19-norpregna-1,3,5(10)-trienes. Synthesis, structures, and cytotoxic, estrogenic, and antiestrogenic effects.

New 3,20-dihydroxy-13α-19-norpregna-1,3,5(10)-trienes were synthesized. The effects of these compounds on breast cancer cells and ERα activation were investigated. The scaffold of compounds containing the six-membered ring D' annulated at 16α,17α-positions was constructed via the Lewis acid catalyzed Diels-Alder reaction of butadiene with 3-methoxy-13α-19-norpregna-1,3,5(10),16-tetraen-20-one 5 under a pressure of 600 MPa. The hydrogenation of primary cyclohexene adduct 6 followed by the one-pot reduction-demethylation (DIBAH) gave target epimeric 3,20-dihydroxy steroids 8a and 8b. The Corey-Chaykovsky reaction of the same conjugated ketone 5 gave a 16α,17α-methylene-substituted compound. The reaction of the latter with DIBAH yielded 3,20(R,S)-dihydroxy-16α,17α-methyleno-13α-19-norpregna-1,3,5(10)-triene 10. The hydrogenation of the 16,17-double bond of compound 5 produced a mixture of 17α- and 17ß-epimeric ketones, reduction-demethylation of which gave 3,20(S)-dihydroxy-13α,17α-19-norpregna-1,3,5(10)-triene 12a and 3,20(R)-dihydroxy-13α,17ß-19-norpregna-1,3,5(10)-triene 12b. All compounds were fully characterized by 1D and 2D NMR, HRMS, and X-ray diffraction. All target compounds showed pronounced cytotoxic effect against MCF-7 breast cancer cells and NCI/ADR-RES doxorubicin-resistant cells at micromolar concentrations. The ERα-mediated luciferase reporter gene assay demonstrated that all compounds, except for compound 10, are ERα inhibitors, while cyclopropane compound 10 proved to be an ERα activator. Docking experiments showed that all compounds are well accommodated to LBD ERα but have some differences in the binding mode.

Amplified Crosstalk Between Estrogen Binding and GFR Signaling Mediated Pathways of ER Activation Drives Responses in Tumors Treated with Endocrine Disruptors.

BACKGROUND: The pharmaceutical development of endocrine disruptors could not achieve appropriate advances in the field of anticancer fight. OBJECTIVE: Considerations on the principles of currently used endocrine therapies. METHODS: Comparison of the results of genetic studies being performed on breast cancer cells treated with estrogens, synthetic estrogens and antiestrogens. RESULTS: In breast cancer cells, increased estrogen concentrations amplify ER-signaling via a synergistic upregulation of both liganded and unliganded ER-activations and increased aromatase expression. The higher the upregulation of ER-signaling, the stronger is the tumor response. Low doses of synthetic estrogens exert an inhibition on the ligand-independent AF1-domain in breast cancer cells, while provoke compensatory activation on the superior, ligand-dependent AF2-domain of ERs and estrogen synthesis. Conversely, high doses of synthetic estrogens induce uncompensated genome-wide disruption in ER-regulated genes leading to toxic symptoms and unpredictable tumor responses. Treatment with antiestrogens, either ER-blockers or aromatase inhibitors, obstructs the crucial AF2-domain of ERs strongly deteriorating the activation of genomic machinery. Tumor responses to antiestrogen treatment depend on the compensatory activation of ER-signaling and the restoration of genomic stability. Recent patents provide methods for the conversion of ER-negative cancers to ER-positive ones improving the possibility of successful treatment. CONCLUSION: In tumor cells, the stabilization of genomic machinery and self-directed death may be achieved via a balanced activation of the AF1 and AF2 domains of ERs by natural estrogen treatment. In contrast, the blockade of either AF1 or AF2 domain by endocrine disruptors leads to toxic symptoms and unforeseeable tumor responses.

Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication.

Hepatitis E virus (HEV) infection causes subclinical diseases, leading to high mortality (>25%) in pregnant women. HEV replication is aggressively escalated in pregnant women, especially in the third trimester of pregnancy. Oestrogen plays an important role in pregnancy. However, the pathogenesis of HEV in pregnant women or immunosuppressive pregnant women (such as HIV-infected or organ-transplanted pregnant women) remains unclear. We investigated the role of oestradiol in HEV infection in a cell culture system. HEV-infected pregnant women had significantly higher oestradiol levels compared with uninfected individuals. HEV infection was significantly increased in cells treated with analogues of oestradiol, diethylstilbestrol (DES) or 17ß-oestradiol in a dose-dependent way. However, tamoxifen, an antagonist oestrogen, inhibited HEV replication. HEV infection inhibits oestrogen receptor (ER-α) expression. Immunofluorescence and co-immunoprecipitation assays indicated that ER-α interacted with the helicase of HEV ORF1 indirectly. More importantly, HEV infection was exacerbated in immunosuppressive cells treated with an inhibitor of PI3K-AKT-mTOR signal pathway (LY296004) and supplemented with pregnant women serum with high oestradiol simultaneously. These results strongly suggest that pregnant women with high oestradiol and/or immunosuppression will be vulnerable to HEV infection.

First-in-Human Phase I Study of the Tamoxifen Metabolite Z-Endoxifen in Women With Endocrine-Refractory Metastatic Breast Cancer.

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor-positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Tamoxifen and its active metabolites inhibit dopamine transporter function independently of the estrogen receptors.

As one of the primary mechanisms by which dopamine signaling is regulated, the dopamine transporter (DAT) is an attractive pharmacological target for the treatment of diseases based in dopaminergic dysfunction. In this work we demonstrate for the first time that the commonly prescribed breast cancer therapeutic tamoxifen and its major metabolites, 4-hydroxytamoxifen and endoxifen, inhibit DAT function. Tamoxifen inhibits [3 H]dopamine uptake into human DAT (hDAT)-N2A cells via an uncompetitive or mixed mechanism. Endoxifen, an active metabolite of tamoxifen, asymmetrically inhibits DAT function in hDAT-N2A cells, showing a preference for the inhibition of amphetamine-stimulated dopamine efflux as compared to dopamine uptake. Importantly, we demonstrate that the effects of tamoxifen and its metabolites on the DAT occur independently of its activity as selective estrogen receptor modulators. This work suggests that tamoxifen is inhibiting DAT function through a previously unidentified mechanism.

Baicalein has protective effects on the 17ß-estradiol-induced transformation of breast epithelial cells.

Epidemiologic and systematic studies have indicated that flavonoid consumption is associated with a lower incidence of breast cancer. Baicalein is the primary flavonoid derived from the roots of Scutellaria baicalensis Georgi. In the current study, the long-term exposure of breast epithelial cells to 17ß-estradiol (E2) was used to investigate the chemopreventive potential of baicalein on neoplastic transformation. The results demonstrated that baicalein significantly inhibited E2-induced cell growth, motility, and invasiveness, and suppressed E2-induced misshapen acini formation in 3D cultures. Furthermore, it inhibited the ability of E2-induced cells to form clones in agarose and tumors in NOD/SCID immunodeficient mice. Docking studies using Sybyl-X 1.2 software showed that baicalein could bind to both estrogen receptor-α (ERa) and G-protein coupled estrogen receptor 30 (GPR30), which are two critical E2-mediated pathways. Baicalein prevented the E2-induced ERa-mediated activation of nuclear transcriptional signaling by interfering with the trafficking of ERa into the nucleus and subsequent binding to estrogen response elements, thereby decreasing the mRNA levels of ERa target genes. It also inhibited E2-induced GPR30-mediated signal transduction, as well as the transcription of GPR30-regulated genes. Therefore, these results suggest that baicalein is a potential drug for reducing the risk of estrogen-dependent breast cancer.

An Antiestrogenic Activity Score for tamoxifen and its metabolites is associated with breast cancer outcome.

PURPOSE: Endoxifen concentrations have been associated with breast cancer recurrence in tamoxifen-treated patients. However, tamoxifen itself and other metabolites also show antiestrogenic anti-tumor activity. Therefore, the aim of this study was to develop a comprehensive Antiestrogenic Activity Score (AAS), which accounts for concentration and antiestrogenic activity of tamoxifen and three metabolites. An association between the AAS and recurrence-free survival was investigated and compared to a previously published threshold for endoxifen concentrations of 5.97 ng/mL. PATIENTS AND METHODS: The antiestrogenic activities of tamoxifen, (Z)-endoxifen, (Z)-4-hydroxytamoxifen, and N-desmethyltamoxifen were determined in a cell proliferation assay. The AAS was determined by calculating the sum of each metabolite concentration multiplied by an IC50 ratio, relative to tamoxifen. The AAS was calculated for 1370 patients with estrogen receptor alpha (ERα)-positive breast cancer. An association between AAS and recurrence was investigated using Cox regression and compared with the 5.97 ng/mL endoxifen threshold using concordance indices. RESULTS: An AAS threshold of 1798 was associated with recurrence-free survival, hazard ratio (HR) 0.67 (95% confidence interval (CI) 0.47-0.96), bias corrected after bootstrap HR 0.69 (95% CI 0.48-0.99). The concordance indices for AAS and endoxifen did not significantly differ; however, using the AAS threshold instead of endoxifen led to different dose recommendations for 5.2% of the patients. CONCLUSIONS: Endoxifen concentrations can serve as a proxy for the antiestrogenic effect of tamoxifen and metabolites. However, for the aggregate effect of tamoxifen and three metabolites, defined by an integrative algorithm, a trend towards improving treatment is seen and moreover, is significantly associated with breast cancer recurrence.

From the Cover: Potentiation of Drug-Induced Phospholipidosis In Vitro through PEGlyated Graphene Oxide as the Nanocarrier.

Cationic amphiphilic drugs (CADs) are small molecules that can induce phospholipidosis (PLD), causing the intracellular accumulation of phospholipid in the lamellar bodies. Nanotechnology based drug delivery systems have been used widely, while it is unknown if drug-induced PLD (DIP) can be potentiated through drug retention by indigestible nanocarriers. Due to the high drug loading capacity of graphene, we investigated if PEGylated graphene oxide (PEG-GO) loaded with CAD could potentiate DIP. Tamoxifen induced the accumulation of NBD-PE, a fluorescence labeled phospholipid in human hepatoma HepG2 cells, while PEG-GO loaded with tamoxifen (PEG-GO/tamoxifen) further potentiated PLD. PEG-GO/tamoxifen induced more gene expression of PLD marker than tamoxifen alone. PEG-GO enhanced DIP was also observed for other CAD, indicating that nanocarrier potentiated DIP could be universal. More lamellar bodies were observed in PEG-GO/tamoxifen treated cells than tamoxifen alone by transmission electron microscopy. When compared with tamoxifen alone, PEG-GO/tamoxifen showed a delayed but potent PLD. In addition, the retarded PLD recovery by PEG-GO/tamoxifen indicated that the reversibility of DIP was interfered. Confocal microscopy revealed the increased number of lysosomes, greater expression of lysosomal associated membrane protein 2 (LAMP2) (a PLD marker), and an increase in the co-localization between lysosome/LAMP2 and NBD-PE by PEG-GO/tamoxifen rather than tamoxifen alone. Finally, we found that PEG-GO or/and tamoxifen-induced PLD seemed to have no correlation with autophagy. This research suggests pharmaceutical companies and regulatory agencies that if nanoparticles are used as the vectors for drug delivery, the adverse drug effects may be further potentiated probably through the long-term accumulation of nanocarriers.