After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Aqueous extracts of 12 Chinese medicinal herbs, Anemarrhena asphodeloides, Artemisia argyi, Commiphora myrrha, Duchesnea indica, Gleditsia sinensis, Ligustrum lucidum, Rheum palmatum, Rubia cordifolia, Salvia chinensis, Scutellaria barbata, Uncaria rhychophylla and Vaccaria segetalis were evaluated for their antiproliferative activity on eight cancer cell lines as well as on normal human mammary epithelial cells. Five human and three murine cancer cell lines representing different tissues (breast, lung, pancreas and prostate) were used. All the crude aqueous extracts demonstrated growth inhibitory activity on some or all of the cancer cell lines, but only two showed activity against the normal mammary epithelial cells. Overall, the murine cell lines tended to be more sensitive to most of the extracts compared with the human cell lines. Among the human cell lines, cell type specificity was observed for two extracts. These results indicate the potential use of traditional Chinese medicinal herbs as antineoplastic agents and suggest that further studies evaluating their mechanism(s) of action and the isolation of active antitumor compounds are warranted.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

BZL 101 inhibits breast cancer cell lines by inducing apoptosis. In a phase I clinical trial, BZL101 was safe and had a favorable toxicity profile. BZL101 demonstrated encouraging clinical activity in this heavily pretreated population.

Estrogen receptor ␤ (ER␤) has potent antiproliferative and anti-inflammatory properties, suggesting that ER␤-selective agonists might be a new class of therapeutic and chemopreventative agents. To understand how ER␤ regulates genes, we identified genes regulated by the unliganded and liganded forms of ER␣ and ER␤ in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ER␣, whereas many genes were regulated by estradiol (E 2 ). These results demonstrated that ER␣ requires a ligand to regulate a single class of genes. In contrast, ER␤ regulated three classes of genes. Class I genes were regulated primarily by unliganded ER␤. Class II genes were regulated only with E 2 , whereas class III genes were regulated by both unliganded ER␤ and E 2 . There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ER␤ regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ER␤ binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFB1, and SP1 sites were more enriched in class II genes. ER␤ bound to all three classes of genes, demonstrating that ER␤ binding is not responsible for differential regulation of genes by unliganded and liganded ER␤. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ER␤ regulate three classes of genes by interacting with different transcription factors and coactivators.Estrogens are essential for the development of the reproductive system. They also exert important actions on nonreproductive tissues such as the bone (1) and cardiovascular system (2). Many biological effects of estrogens are mediated by two estrogen receptors (ERs), 4 estrogen receptor ␣ (ER␣; NR3A1 and ESR1) and estrogen receptor ␤ (ER␤; NR3A2 and ESR2) (3, 4).

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions . In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0 .1 to 0.2 g/min per cm2 (initial cross-sectional area) . Electron microscopy of clots fixed after attaining a force of 1 .6 g/cm 2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension . The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction . Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i .e . fibrin) . Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed . Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands . Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation . Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract . The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension . The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers .The phenomenon of clot retraction has been recognized for more than two hundred years (20). Physiologically, clot retraction may enhance clot lysis and facilitate recanalization of blood vessels (6) . Mechanistically, clot contraction remains poorly understood, though in platelet-rich plasma it has been clearly shown to result from the interaction of a static, nonmotile fibrin mesh and dynamic, actively motile blood platelets (5,12,13,29,39) . Concepts concerning interaction of these two components can be classed into three general proposals . The first suggests that platelets reach out with their pseudopods to combine with and attach to strands of fibrin. Pseudopods then contract, pulling the fibrin mesh toward the platelets . According to one variation of this concept, the center of the platelet may be an anchoring point for pulling in the fibers (35,43) . Another modification proposes that contraction is related to THE JOURNAL OF CELL BIOLOGY " VOLUME 93 JU...

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERβ. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by nonselectively triggering both ERα and ERβ pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERβ should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERβ regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα-and ERβ-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERβ agonists are promising new approach for treating specific conditions associated with menopause.

Genome sequencing has established clinical utility for rare disease diagnosis. While increasing numbers of individuals have undergone elective genome sequencing, a comprehensive study surveying genome-wide disease-associated genes in adults with deep phenotyping has not been reported. Here we report the results of a 3-y precision medicine study with a goal to integrate wholegenome sequencing with deep phenotyping. A cohort of 1,190 adult participants (402 female [33.8%]; mean age, 54 y [range 20 to 89+]; 70.6% European) had whole-genome sequencing, and were deeply phenotyped using metabolomics, advanced imaging, and clinical laboratory tests in addition to family/medical history. Of 1,190 adults, 206 (17.3%) had at least 1 genetic variant with pathogenic (P) or likely pathogenic (LP) assessment that suggests a predisposition of genetic risk. A multidisciplinary clinical team reviewed all reportable findings for the assessment of genotype and phenotype associations, and 137 (11.5%) had genotype and phenotype associations. A high percentage of genotype and phenotype associations (>75%) was observed for dyslipidemia (n = 24), cardiomyopathy, arrhythmia, and other cardiac diseases (n = 42), and diabetes and endocrine diseases (n = 17). A lack of genotype and phenotype associations, a potential burden for patient care, was observed in 69 (5.8%) individuals with P/LP variants. Genomics and metabolomics associations identified 61 (5.1%) heterozygotes with phenotype manifestations affecting serum metabolite levels in amino acid, lipid and cofactor, and vitamin pathways. Our descriptive analysis provides results on the integration of whole-genome sequencing and deep phenotyping for clinical assessments in adults.genomics | advanced imaging | precision medicine | deep phenotyping | metabolomics