Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/ß Ratio Alteration and ROS Production.

BACKGROUND: Low risk of breast cancer is observed among females consuming a moderate quantity of soy throughout their life. The present study was conducted to evaluate the anticancer potential of Daidzein, one of the major Isoflavones in soy using Human breast cancer cells MCF-7. METHODS: MCF-7 were subjected to various doses of Daidzein treatment to determine the IC50 value. Onset of apoptosis was ascertained by AnnexinV assay and caspase 3/7 activity post treatment. Expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 was also assessed to further confirm apoptotic mode of cell death. ROS production post treatment with Daidzein was assessed to ascertain the apoptosis via intrinsic pathway. Expression of ER α and ER ß was evaluated by western blot analysis. RESULTS: Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC50 value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER ß has been observed which are conducive indicator of apoptosis. CONCLUSIONS: In conclusion, the present study suggests that Daidzein induces apoptosis in MCF-7 cells by mitochondrial pathway along with lowering the ratio of ER α/ß and an outburst of Reactive Oxygen Species(ROS).

Estrogenic phytochemical from Labisia pumila (Myrsinaceae) with selectivity towards estrogen receptor alpha and beta subtypes.

Labisia pumila var. alata (Myrsinaceae) or "Kacip fatimah" is a famous Malay traditional herb used for the maintenance of women's health. The extracts of L.pumila displayed estrogenic activity in rats. Nonetheless, the estrogenic bioactives were not identified. The aim of the study is to identify estrogenic compounds contributing to the established estrogenic activity. Bioactivity-guided-isolation method guided the isolation of pure bioactives. The hexane extract was subjected to a series of silica gel flash and open column chromatography with increasing amount of ethyl acetate in hexane or methanol in chloroform. Each fraction or pure compounds were evaluated on it's estrogen receptor (ER) binding activity with the fluorescence polarization competitive ERα and ERß binding assay kit. Cytotoxic assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method was used to establish the cytotoxic activity of the compounds. Four alkyl resorcinols and a dimeric 1,4-benzoquinone, namely belamcandol B (1), 5-pentadec-10'-(Z)-enyl resorcinol (2), 1,3-dihydroxy-5-pentadecylbenzene (3), 5-(heptadec-12'-(Z)-enyl) resorcinol (4) and demethylbelamcandaquinone B (5) were identified with selective binding affinities towards either ERα or ERß exhibiting selectivity ratio from 0.15-11.9. Alkyl resorcinols (2)-(4) exhibited cytotoxic activity towards HL60 cells with IC50 values from 19.5-22.0 µM. Structural differences between compounds influence the binding affinities to ER subtypes. Further study is needed to establish the agonist or antagonist effect of these compounds on various tissues and to identify if these compounds exert cytotoxic activity through the ERs. When consuming L.pumila as a complementary medicine, careful consideration regarding it's estrogenic compound content should be given due consideration.

Selected Phytoestrogens Distinguish Roles of ERα Transactivation and Ligand Binding for Anti-Inflammatory Activity.

Estrogen receptor α (ERα) is a ligand-activated transcriptional activator that is also involved vascular inflammation and atherosclerosis. Whether different ligands may affect this activity has not been explored. We screened a panel of phytoestrogens for their role in ERα binding and transcriptional transcription, and correlated the findings to anti-inflammatory activities in vascular endothelial cells stably expressing either a wild-type or mutant form of ERα deficient in its membrane association. Taxifolin and silymarin were "high binders" for ERα ligand binding; quercetin and curcumin were "high activators" for ERα transactivation. Using these phytoestrogens as functional probes, we found, in endothelial cells expressing wild-type ERα, the ERα high activator, but not the ERα high binder, promoted ERα nuclear translocation, estrogen response element (ERE) reporter activity, and the downstream gene expression. In endothelial cells expressing membrane association-deficient mutant ERα, the ERα nuclear translocation was significantly enhanced by taxifolin and silymarin, which still failed to activate ERα. Inflammation response was examined using the systemic or vascular inflammation inducers lipopolysaccharide or oxidized low-density lipoprotein. In both cases, only the ERα high activator inhibited nuclear translocation of nuclear factor κB, JNK, and p38, and the production of inflammatory cytokines IL-1ß and TNFα. We confirm a threshold nuclear accumulation of ERα is necessary for its transactivation. The anti-inflammatory activity of phytoestrogens is highly dependent on ERα transactivation, less so on the ligand binding, and independent of its membrane association. A pre-examination of phytoestrogens for their mode of ERα interaction could facilitate their development as better targeted receptor modifiers.

Arctigenin inhibits the activation of the mTOR pathway, resulting in autophagic cell death and decreased ER expression in ER-positive human breast cancer cells.

Arctigenin, a member of the Asteraceae family, is a biologically active lignan that is consumed worldwide due to its several health benefits. However, its use may pose a problem for patients with estrogen receptor (ER)α-positive breast cancer, since studies have shown that arctigenin is a phytoestrogen that exerts a proliferative effect by binding to the ER. Thus, in this study, we examined the effect of arctigenin on ERα-positive MCF-7 human breast cancer cells to determine whether the consumption of arctigenin is safe for patients with breast cancer. First, we found that arctigenin inhibited the viability of the MCF-7 cells, and colony formation assay confirmed that this effect was cytotoxic rather than cytostatic. The cytotoxic effects were not mediated by cell cycle arrest, apoptosis, or necroptosis, despite DNA damage, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and phosphorylated H2A.X. An increase in lipidated LC3, a marker of autophagosome formation, was observed, indicating that autophagy was induced by arctigenin, which was found to be triggered by the inhibition of the mechanistic target of rapamycin (mTOR) pathway. We then examined the effects of arctigenin on ERα expression and determined whether it affects the sensitivity of the cells to tamoxifen, as tamoxifen is commonly used against hormone-responsive cancers and is known to act via the ERα. We found that treatment with arctigenin effectively downregulated ERα expression, which was found to be a consequence of the inhibition of the mTOR pathway. However, treatment with arctigenin in combination with tamoxifen did not affect the sensitivity of the cells to tamoxifen, but instead, exerted a synergistic effect. On the whole, our data indicate that the phytoestrogen, arctigenin, mainly targeted the mTOR pathway in ERα-positive MCF-7 human breast cancer cells, leading to autophagy-induced cell death and the downregulation of ERα expression. Furthermore, the synergistic effects between arctigenin and tamoxifen suggest that the consumption of arctigenin is not only safe for patients with hormone-sensitive cancers, but may also be an effective co-treatment.

Sex Differences in Steroid Receptor Coexpression and Circadian-Timed Activation of Kisspeptin and RFRP-3 Neurons May Contribute to the Sexually Dimorphic Basis of the LH Surge.

In rodents, the ovulation-inducing luteinizing hormone (LH) surge is sexually dimorphic, occurring only in females, but the reasons for this sex difference are unclear. Two neuropeptides, kisspeptin and RFamide-related peptide 3 (RFRP-3), are hypothesized to regulate the gonadotropin-releasing hormone (GnRH)/LH surge. In females, both of these systems show circadian changes coincident with the LH surge, but whether males show similar temporal changes under comparable hormonal conditions is unknown. Here, we evaluated circadian time (CT)-dependent changes in gene expression and neuronal activation of Kiss1 and Rfrp neurons of female and male mice given identical LH surge-inducing estrogen regimens. As expected, females, but not males, displayed a late afternoon LH surge and GnRH neuronal activation. Kiss1 expression in the anteroventral periventricular nucleus (AVPV) was temporally increased in females in the late afternoon, whereas males demonstrated no temporal changes in AVPV Kiss1 expression. Likewise, neuronal activation of AVPV Kiss1 neurons was dramatically elevated in the late afternoon in females but was low at all circadian times in males. Estrogen receptor α levels in AVPV Kiss1 neurons were sexually dimorphic, being higher in females than males. AVPV progesterone receptor levels were also higher in females than males. Hypothalamic Rfrp messenger RNA levels showed no CT-dependent changes in either sex. However, Rfrp neuronal activation was temporally diminished in the afternoon/evening in females but not males. Collectively, the identified sex differences in absolute and CT-dependent AVPV Kiss1 levels, AVPV sex steroid receptor levels, and circadian-timed changes in neuronal activation of both Kiss1 and Rfrp neurons suggest that multiple sexually dimorphic processes in the brain may underlie proper LH surge generation.

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation.

Estrogen receptors (ERs) α and ß are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as a model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases.

Genistein disrupts glucocorticoid receptor signaling in human uterine endometrial Ishikawa cells.

BACKGROUND: The link between environmental estrogen exposure and defects in the female reproductive tract is well established. The phytoestrogen genistein is able to modulate uterine estrogen receptor (ER) activity, and dietary exposure is associated with uterine pathologies. Regulation of stress and immune functions by the glucocorticoid receptor (GR) is also an integral part of maintaining reproductive tract function; disruption of GR signaling by genistein may also have a role in the adverse effects of genistein. OBJECTIVE: We evaluated the transcriptional response to genistein in Ishikawa cells and investigated the effects of genistein on GR-mediated target genes. METHODS: We used Ishikawa cells as a model system to identify novel targets of genistein and the synthetic glucocorticoid dexamethasone through whole genome microarray analysis. Common gene targets were defined and response patterns verified by quantitative real-time reverse-transcription polymerase chain reaction. The mechanism of transcriptional antagonism was determined for select genes. RESULTS: Genistein regulated numerous genes in Ishikawa cells independently of estradiol, and the response to coadministration of genistein and dexamethasone was unique compared with the response to either estradiol or dexamethasone alone. Furthermore, genistein altered glucocorticoid regulation of GR target genes. In a select set of genes, co-regulation by dexamethasone and genistein was found to require both GR and ERα signaling, respectively. CONCLUSIONS: Using Ishikawa cells, we observed that exposure to genistein resulted in distinct changes in gene expression and unique differences in the GR transcriptome.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

An alternative transcription start site yields estrogen-unresponsive Kiss1 mRNA transcripts in the hypothalamus of prepubertal female rats.

The importance of the Kiss1 gene in the control of reproductive development is well documented. However, much less is known about the transcriptional regulation of Kiss1 expression in the hypothalamus. Critical for these studies is an accurate identification of the site(s) where Kiss1 transcription is initiated. Employing 5'-RACE PCR, we detected a transcription start site (TSS1) used by the hypothalamus of rats, mice, nonhuman primates and humans to initiate Kiss1 transcription. In rodents, an exon 1 encoding 5'-untranslated sequences is followed by an alternatively spliced second exon, which encodes 5'-untranslated regions of two different lengths and contains the translation initiation codon (ATG). In nonhuman primates and humans, exon 2 is not alternatively spliced. Surprisingly, in rat mediobasal hypothalamus (MBH), but not preoptic area (POA), an additional TSS (TSS2) located upstream from TSS1 generates an exon 1 longer (377 bp) than the TSS1-derived exon 1 (98 bp). The content of TSS1-derived transcripts increased at puberty in the POA and MBH of female rats. It also increased in the MBH after ovariectomy, and this change was prevented by estrogen. In contrast, no such changes in TSS2-derived transcript abundance were detected. Promoter assays showed that the proximal TSS1 promoter is much more active than the putative TSS2 promoter, and that only the TSS1 promoter is regulated by estrogen. These differences appear to be related to the presence of a TATA box and binding sites for transcription factors activating transcription and interacting with estrogen receptor-α in the TSS1, but not TSS2, promoter.

Resveratrol and endometrium: a closer look at an active ingredient of red wine using in vivo and in vitro models.

Resveratrol is a natural phytoestrogen with antiproliferative properties present in red wine, grapes, and berries. Published reports on the effects of resveratrol in human endometrial function are limited. The objective of this study was to investigate the expression of estrogen receptor α (ESR1), Ki-67 (a proliferative marker), aryl hydrocarbon receptor (AhR), and members of the cytochrome P450 superfamily of enzymes (CYP1A1 and CYP1B1) in an in vitro and vivo assay. Alkaline phosphatase assay of estrogenicity was used to compare estrogen activity of different concentrations of resveratrol to estradiol (E2) and diethylstilbestrol (DES), using Ishikawa cell culture. Immunohistochemical expression of ESR1 and Ki67, and reverse transcriptase polymerase chain reaction of AhR, CYP1A1, and CYP1B1 were analyzed from xenograft implants of human endometrial tissue in ovariectomized immunodeficient RAG-2-γ(c) mice, after 30 days of treatment with subcutaneous pellets of E2, E2 plus progesterone (P4), or E2 plus resveratrol (6, 30, or 60 mg) for 30 days. Compared to E2, resveratrol acted as an agonist and antagonist of estrogen in low and high concentrations, respectively, when combined with E2. Xenografts of human endometrial tissues in RAG-2 mice exhibited reduced expression of ESR1 and proliferative activity (Ki67) with 60 mg of resveratrol. This study suggests that resveratrol, at high doses, has the potential benefit to reduce proliferation of human endometrium through ESR1.

Neonatal testosterone suppresses a neuroendocrine pulse generator required for reproduction.

The pituitary gland releases hormones in a pulsatile fashion guaranteeing signalling efficiency. The determinants of pulsatility are poorly circumscribed. Here we show in magnocellular hypothalamo-neurohypophyseal oxytocin (OT) neurons that the bursting activity underlying the neurohormonal pulses necessary for parturition and the milk-ejection reflex is entirely driven by a female-specific central pattern generator (CPG). Surprisingly, this CPG is active in both male and female neonates, but is inactivated in males after the first week of life. CPG activity can be restored in males by orchidectomy or silenced in females by exogenous testosterone. This steroid effect is aromatase and caspase dependent, and is mediated via oestrogen receptor-α. This indicates the apoptosis of the CPG network during hypothalamic sexual differentiation, explaining why OT neurons do not burst in adult males. This supports the view that stereotypic neuroendocrine pulsatility is governed by CPGs, some of which are subjected to gender-specific perinatal programming.

Impact of lycopene on epididymal androgen and estrogen receptors' expression in polychlorinated biphenyls-exposed rat.

The aim of the study was to evaluate the androgen (AR) and estrogen receptors' (ER) expression in epididymis of polychlorinated biphenyls (PCBs)-exposed rats. The rats were assigned to groups. Group I controls were treated with corn oil 80 µL/d intraperitoneally (ip), group II were treated with 2 mg/kg/d of A1254 ip; and group III were treated with 2 mg/kg/d of A1254 ip along with simultaneous oral supplementation of 4 mg/kg/d lycopene . The treatment was given daily for 30 days. After 24 hours of treatment, the rats were killed, and the epididymal regions (caput, corpus, and cauda) were dissected out, weighed, and prepared to estimate the levels of sialic acid, glyceryl phosphoryl choline (GPC), hydrogen peroxide (H2O2), and lipid peroxidation (LPO). The messenger RNA (mRNA) expressions of AR, ERα, and ERß were analyzed by reverse transcriptase-polymerase chain reaction, and ERα and ERß protein expressions were analyzed by immunoblotting. The toxicity of PCBs was also confirmed by histology. There was a marked decrease in epididymal weight, sialic acid, and GPC levels, while oxidative stress markers H2O2 and LPO were increased in PCBs-treated rats. The mRNA and protein expression of AR, ERα, and ERß were decreased in PCBs-treated groups, and the histology confirms the cytoplasmic damage in the regions of caput, corpus, and cauda in PCBs-treated rats. Simultaneous supplementation of lycopene to PCBs-exposed rats resulted in significant decrease in the oxidative stress markers as that of control, while the AR, ERα, and ERß gene expressions were near to control. The results suggest that lycopene has ameliorative effect against PCBs-induced toxicity in epididymis.

Estrogen receptor mediated effects of Cimicifuga extracts on human breast cancer cells.

Cimicifuga racemosa extracts have long been used to treat female reproductive disorders both in Asia and Europe. Here in this study, we examined the possible estrogen receptor (ER)alpha effects of Cimicifuga heracleifolia var. bifida ethanol extract (C-Ex), which has been used traditionally in Asia, in MCF-7 cells. The activity of C-Ex was characterized in a transient transfection system, using ERa and estrogen-responsive luciferase plasmids in HEK 293 cells and endogenous target genes were studied in MCF-7 cells. C-Ex failed to activate ERalpha and at a concentration of 0.005-0.5 mg/ml as examined by reporter activity. In addition, no statistically significant antiestrogenic activity was observed. However, to our interest, C-Ex enhanced expression of VEGF at 0.5 mg/ml concentration and repressed ERalpha both at the mRNA and protein levels in MCF-7 cells. These results suggested that C-Ex does not activate or inactivate ERalpha in a direct manner, but the extracts may affect factors in ER signal transduction pathway.

Effects of bazedoxifene acetate with and without conjugated equine estrogens on the breast of postmenopausal monkeys.

OBJECTIVE: Concerns about increased breast cancer risk with estrogen and progestin therapy have led to an increased interest in progestin alternatives. The main objective of this study was to determine if bazedoxifene acetate (BZA), a new selective estrogen receptor modulator, will antagonize the proliferative and transcriptional effects of conjugated equine estrogens (CEE) in the breast. METHODS: As part of a 20-month preclinical trial, 95 ovariectomized cynomolgus macaques (Macaca fascicularis) were randomized to receive no treatment or treatment with BZA (20 mg/d), CEE (0.45 mg/d), or BZA and CEE in combination (women's daily equivalent doses). The data presented here include breast effects after 6 months of treatment. Endpoints included histomorphometry, histopathological evaluations, gene microarray assays, polymerase chain reaction quantification of specific estrogen receptor α (ER-α) activity markers, and immunohistochemical detection of sex steroid receptors, and the proliferation marker Ki67. RESULTS: BZA + CEE and BZA resulted in significantly less total epithelial density, lobular enlargement, and Ki67 immunolabeling in the terminal ducts compared with CEE alone (P < 0.05 for all). The addition of BZA to CEE antagonized the expression of ER-α-regulated genes such as GREB1 and TFF1 (P < 0.01 for both), whereas BZA alone had minimal effects on ER-α-mediated transcriptional activity. BZA and BZA + CEE did not significantly up-regulate genes related to cell cycle progression and proliferation. BZA with and without CEE also resulted in less lobular and terminal duct ER-α immunolabeling compared with control and CEE (P < 0.0001 for all). CONCLUSIONS: These findings demonstrate that BZA given at a clinically relevant dose is an estrogen antagonist in the breast, supporting the idea that CEE + BZA may provide a lower breast cancer risk profile compared with traditional estrogen + progestin therapies.

The rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure.

Current knowledge about dietary soy isoflavone-induced hormonal effects and potential priming effects for the responsiveness of the organism to other estrogens is insufficient. The present study examined the effects of pre- and postnatal soy isoflavone exposure on estrogen responsiveness by estrogen receptor agonists in the uteri of prepubertal Wistar rats. To this end, offspring were generated from dams already maintained on three dietary groups, (1) a phytoestrogen-free diet, (2) a soy isoflavone-rich diet with 232 ppm daidzein and 240 ppm genistein or (3) a custom-made diet supplemented with 700 ppm genistein (GEN). Then, F1 females continuously exposed to isoflavones from GD1 to PND21 and non-exposed controls were subjected to an immature uterotrophic assay to compare physiological parameters and the response to subcutaneous treatment with 17ß-estradiol, GEN or an estrogen receptor subtype (ERα and ERß)-specific agonist. Uterine wet weight (UWW), luminal epithelial height (LEH) and myometrial thickness (MMT) were determined. In addition, isoflavone plasma levels and mRNA expression profiles of relevant steroid receptors and of molecular markers for proliferation and estrogenicity were assessed for all groups. The influence of dietary isoflavones on the sensitivity to various estrogenic stimuli in these prepubertal animals was minor. Yet, the uterus of immature rats with high chronic GEN exposure alone showed already an increase in UWW, LEH and MMT. The myometrial response to GEN was more pronounced than that of the luminal epithelium, which may be due to a non-uniform distribution of steroid receptors, in particular the progesterone receptor. In conclusion, although the impact of a continuous, prenatally initiated exposure to dietary isoflavones on the uterine physiology of juvenile rats is modest, the possible priming effects of this exposure for beneficial or adverse late-onset consequences in adults should not be neglected.

Selective estrogen receptor modulators regulate stromal proliferation in human benign prostatic hyperplasia by multiple beneficial mechanisms--action of two new agents.

The existing drugs for benign prostatic hyperplasia (BPH) are partially effective with undesirable side-effects; hence new agents acting by different mechanism(s) are required as supplements. Modulation of estrogen receptor signaling using selective estrogen receptor modulators (SERMs) offers an alternative approach for BPH management. Using human BPH-derived stromal cells and tissue explants in culture we evaluated two SERMs, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2 H-1-benzopyran (BP) and Ormeloxifene (Orm) in comparison to Tamoxifen (Tam) and 4-hydroxytamoxifen (OHT). BP, OHT and Tam were more effective than Orm in reducing stromal cell proliferation of human BPH. BP was either equipotent or more effective than OHT and Tam in increasing estrogen receptor(ER)-ß, TGFß1, Fas and FasL, and in decreasing ER-α, AR, EGF-R and IGF-I expressions in BPH stromal cells. BP, Tam and Orm (1.0 mg/Kg) reduced rat prostate weights by almost same extent as Finasteride (Fin, 5.0 mg/Kg); however combination treatment (SERM+Fin) was more effective. BP was exceptionally efficient in reducing IGF-1 and cleaving PARP while combination treatments more effectively increased bax:bcl-2 ratio. Fin reduced acinar diameter and prostatic DHT level but increased testosterone, estradiol (E(2)) and E(2)/T+DHT ratio. SERMs, especially BP, reduced epithelial cell height drastically without significantly altering steroid hormone levels and E(2)/T+DHT ratio. Combination treatment reduced both acinar diameter and epithelial cell height with modest increase in E(2), T and E(2)/T+DHT. The study reveals the potential of SERMs per se for BPH management, and more effectively in combination with a 5α-reductase inhibitor. BP appears promising for further evaluation as a drug candidate for BPH and prostate cancer.

Reproductive toxicity of Rhizoma Sparganii (Sparganium stoloniferum Buch.-Ham.) in mice: mechanisms of anti-angiogenesis and anti-estrogen pharmacologic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Indications and preliminary studies of Rhizoma Sparganii (RS) suggest its pharmacological mechanism is involved with endocrine/angiogenesis functions. We therefore studied its potential toxicity on reproduction in mice. MATERIALS AND METHODS: Reproductive toxicity of 100, 200 and 400 mg/kg RS extract were studied in pregnant ICR mice and its offspring. The embryos' fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor (VEGF) and estrogen receptor-α (ER-α) were evaluated as targets of endocrine/angiogenesis by immunohistochemical test. RESULTS: The offspring of treated mice (100, 200 and 400 mg/kg RS extract) during their pregnancy had various pathological conditions, suggesting an abnormal FGF signaling phenomenon during pregnancy. Embryos from the 400 mg/kg group had significantly depressed levels of FGF-1 (P < 0.01) and VEGF (P < 0.05) expression levels as compared to controls by immunohistochemical test. Dysplasia in the heart (12.9%), craniofacial region (18.3%) and vertebrae (32.5%) presented in embryos of the 400 mg/kg group. Furthermore, the ER-α expression was inversely proportional to FGF-1 levels in the same embryo (P < 0.01). CONCLUSIONS: These results implicate a FGF signaling abnormality in vivo and indicate that RS has anti-angiogenesis and anti-estrogen toxicity effects in pregnant rodents.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. MATERIALS AND METHODS: The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. RESULTS: The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of ß-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, ß-naphthoflavone (ßNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by ßNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. CONCLUSION: The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Calycosin protects HUVECs from advanced glycation end products-induced macrophage infiltration.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali radix is a traditional Chinese medicine that has long been used for treatment of diabetes and diabetes-associated disease, but its active component and mechanism on the disease is not well defined. AIM OF THE STUDY: Infiltration of leukocytes within the glomeruli and vasculature is one of the early and characteristic features of diabetic nephropathy. Advanced glycation end products (AGEs) play pivotal role in the progression of diabetic-associated diseases. The present study was designed to explore the therapeutic effect of calycosin, an active component from A. radix, on AGEs-induced macrophages infiltration in HUVECs. MATERIALS AND METHODS: Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions; real-time PCR was carried out to determine mRNA expression of TGF-beta1, ICAM-1 and RAGE. Immunofluorescence was carried out to observe estrogen receptor-alpha, ICAM-1, RAGE expression and the phosphorylation status of ERK1/2 and NF-κB. RESULTS: Calycosin significantly reduced AGEs-induced macrophage migration and adhesion to HUVEC. Pre-treatment with calycosin strikingly down-regulated HUVEC TGF-beta1, ICAM-1 and RAGE expressions in both protein and mRNA levels. Furthermore, calycosin incubation significantly increased estrogen receptor expression and reversed AGEs-induced ERK1/2 and NF-κB phosphorylation and nuclear translocation in HUVEC, and this effect of calycosin could be inhibited by estrogen receptor inhibitor, ICI182780. CONCLUSIONS: These findings suggest that calycosin can reduce AGEs-induced macrophage migration and adhesion to endothelial cells and relieve the local inflammation; furthermore, this effect was via estrogen receptor-ERK1/2-NF-κB pathway.

Estrogenic activity of chemical constituents from Tephrosia candida.

In a continued investigation of medicinal plants from the genus Tephrosia, phytochemical analysis of a methylene chloride-methanol (1:1) extract of the air-dried aerial parts of Tephrosia candida afforded two new 8-prenylated flavonoids, namely, tephrocandidins A (1) and B (2), a new prenylated chalcone, candidachalcone (3), a new sesquiterpene (4), and a previously reported pea flavonoid phytoalexin, pisatin (5). The structures of 1-4 were established by spectroscopic methods, including HREIMS, and 1H, 13C, DEPT, HMQC, and HMBC NMR experiments. The most potent estrogenic activity of these isolated natural products in an estrogen receptor (ERα) competitive-binding assay was for 3, which exhibited an IC50 value of 80 µM, compared with 18 nM for the natural steroid 17ß-estradiol. Results were interpreted via virtual docking of isolated compounds to an ERα crystal structure.