Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice.

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER(-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor in the BE group and at pre-tumor and post-tumor in the LC group. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia, Lactobacillus genus and Lachnospiraceae, S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER(-) breast cancer prevention.

ER-depletion lowering the 'hypothalamus-uterus-kidney' axis functions by perturbing the renal ERß/Ptgds signalling pathway.

Researchers have long assumed that systematic estrogen fading might contribute to the sustained progression of menopausal degenerate syndromes, although definitive evidence has not been presented. Whether such findings represent a causal contribution or are the result of opportunistic messengers sent from the reproductive system to the brain is also a vital question. We constructed a multiscale network of the ovariectomy (OVX) induced estrogen receptors depletion (ER-depletion) model and integrated targeted proteomic, targeted lipidomic, cytochemical, and histopathological data across three tissues from the ovariectomy rodent model. We found that compared to control rats, OVX rats showed increased renal and uterine prostaglandin D2 synthase (Ptgds) expression and decreased hypothalamic Ptgds expression, abnormal Ptgds metabolites, the degenerate renal function profiles and decreased cognitive ability (learning and memory) in Morris water maze test. Importantly, we observed a regulatory relationship among ER (particularly ERß), the degree of the pathological phenotype, learning behavior test and the 'hypothalamus-uterus-kidney (HUK) axis functions. Collectively, this study elucidates that ER depletion promoted HUK aging is mostly attributed to a renal ERß/Ptgds signalling imbalance.

Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation.

This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation.

Estrogenic effects of fusarielins in human breast cancer cell lines.

The fusarielins are a group of metabolites found in several Aspergillus and Fusarium species that have been reported to have with weak antifungal, antibiotic and cytotoxic effects. This study identifies fusarielin A, F, G and H isolated from Fusarium as mycoestrogens. Mycoestrogens are compounds from fungi that bind to the estrogen receptors and induce an estrogenic response in targeted cells. All four tested fusarielins stimulate MCF-7 cell proliferation with fusarielin H as the most potent, able to stimulate cell proliferation 4-fold in a resazurin metabolism assay at 25µM. MDA-MB-231 cells without the estrogen receptor-α and MCF-10a cells without estrogen receptors were not stimulated by fusarielins. Furthermore, the stimulation was prevented in MCF-7 cells when fusarielins were incubated in the presence of the estrogen receptor antagonist fulvestrant. These observations suggest that fusarielins bind to the estrogen receptor and act as weak mycoestrogens.

Photo-activated pheophorbide-a, an active component of Scutellaria barbata, enhances apoptosis via the suppression of ERK-mediated autophagy in the estrogen receptor-negative human breast adenocarcinoma cells MDA-MB-231.

AIM OF THE STUDY: Scutellaria barbata is a traditional Chinese medicine for cancer treatments. Pheophorbide-a (Pa), one of the active components isolated from this herbal medicine has been proposed to be a potential natural photosensitizer for photodynamic therapy. The anti-tumor effect of pheophorbide-a based photodynamic therapy (Pa-PDT) has been successfully demonstrated in a wide range of human malignant cell lines. However, the effectiveness of Pa-PDT has not yet been evaluated on human breast cancer, which is documented as the second common and the fifth most lethal cancer worldwide. MATERIALS AND METHODS: The cytotoxicity of Pa-PDT was evaluated by using an estrogen receptor (ER)-negative human breast adenocarcinoma cell line MDA-MB-231. The involvement of mitochondria was revealed by the change of mitochondrial membrane potential and the increase of intracellular reactive oxygen species (ROS). The hallmarks of apoptosis, ER stress and autophagy were also assessed by DNA fragmentation, Western blotting, and immunostaining assays. RESULTS: Pa-PDT showed inhibitory effect on the growth of MDA-MB-231 cells with an IC(50) value of 0.5 microM at 24h. Mitogen-activated protein kinase (MAPK) pathway was found to be triggered, where activation of c-Jun N-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK) were occurred in the Pa-PDT-treated cells. Our findings suggested that Pa-PDT exhibited its anti-tumor effects by the activation of mitochondria-mediated apoptosis and the ERK-mediated autophagy in MDA-MB-231 cells. CONCLUSION: The present study suggested Pa-PDT is a potential protocol for the late phase human breast cancer, and it is the first study to demonstrate the Pa-PDT induced autophagy contributed to the anti-tumor effects of Pa-PDT on human cancer cells.

Grape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptor-negative MDA-MB231 tumors in nu/nu mouse xenografts.

The antitumor properties of the Merlot grape (and Merlot wine) polyphenols were evaluated in relation to their ability to modulate gene expression in developing tumors using an athymic nude mouse model transplanted with the estrogen receptor-negative MDA-MB231 cells. Groups of mice were fed a modified AIN 93G diet (Research Diets Inc, New Brunswick, NJ) with the experimental groups receiving 100 mg/kg body weight equivalent of polyphenols by gavage 3 times per week. After 1 week of acclimation and another week of polyphenol supplementation, MDA-MB231 cells were transplanted and the growth patterns of the tumors monitored. After 33 days of tumor growth, the animals were euthanized, the tumors isolated, and gene expression profiles analyzed using signal transduction and cell cycle arrays. The development of tumors was almost totally arrested in grape polyphenol-treated mice. Total polyphenols isolated from the wine were more effective in reducing tumor growth as compared with a hydrophobic polyphenol fraction isolated from the wine, showing a 50% and 60% reduction in tumor growth on day 33, respectively. Analysis of gene expression showed that genes such as CDK2, FAS, LEF1, PRKCE, and PTGS2, belonging to the NFkappaB, phospholipase C, and calcium signaling pathways, were down-regulated in tumors that developed in grape polyphenol-treated mice. Several genes related to cell cycle regulation, such as CDK5RAP1, RBBP8, and SERTAD1, were up-regulated in these tumors. Changes in the expression of these genes were less pronounced in tumors of wine polyphenol-treated mice. The study highlights the potential influences of dietary polyphenolic components on gene expression in estrogen receptor-negative tumors and its relation to inhibition of tumor growth.

Agents with selective estrogen receptor (ER) modulator activity induce apoptosis in vitro and in vivo in ER-negative glioma cells.

Tamoxifen, a member of the selective estrogen receptor modulator (SERM) family, is widely used in the treatment of estrogen receptor (ER)-expressing breast cancer. It has previously been shown that high-dose tamoxifen has cytotoxic activity against glioma cells, but whether this effect is drug specific or represents a general property of SERMs is unknown. In this study, we demonstrate that tamoxifen and CC-8490, a novel benzopyranone with SERM activity, induce glioma cell apoptosis in a dose- and time-dependent manner. Moreover, administration of tamoxifen and CC-8490 suppresses tumor growth in vivo and extends animal survival in glioma xenograft models. None of the eight glioma cell lines examined express either ER-alpha or -beta, suggesting the mechanism for tamoxifen- and CC-8490-induced glioma cell apoptosis is independent of the ER signaling pathway. Complementary DNA microarray expression profiling allowed us to identify a subset of genes specifically regulated by tamoxifen and CC-8490, and not by other apoptotic stimuli, including nuclear factor (NF)-kappaB with its target genes IEX-3, SOD2, IL6, and IL8. We demonstrate that suppression of NF-kappaB activation markedly enhances SERM-induced apoptosis, suggesting a role for NF-kappaB in protecting glioma cells from SERM-induced cytotoxicity. These findings demonstrate for the first time that a SERM other than tamoxifen can induce glioma cell apoptosis in vitro and in vivo and that the clinical efficacy of SERMs for the treatment of malignant gliomas could potentially be enhanced by simultaneous inhibition of the NF-kappaB pathway.

Octamethylcyclotetrasiloxane exhibits estrogenic activity in mice via ERalpha.

Octamethylcyclotetrasiloxane (D4) is a low molecular weight cyclic silicone used in the synthesis of larger silicone polymers and in the formulation of a variety of personal care products. The effects of oral D4 exposure in mice on serum estradiol levels, uterine wet weight, and uterine peroxidase activity were investigated. Additionally, in vitro estrogen receptor binding activity was evaluated. Serum estradiol levels decreased in a dose-dependent manner after exposure to 100 mg/kg to 1000 mg/kg D4. Studies with adrenalectomized animals demonstrated that the decreased serum estradiol levels were not due to elevated serum corticosterone levels. Uterine wet weights in ovariectomized mice were significantly increased in a dose-dependent manner by exposure to 250-1000 mg of D4/kg, but not by exposure to other silicone compounds tested (hexamethylcyclotrisiloxane, decamethylcyclopentasiloxane, decamethyltetrasiloxane, and octaphenylcyclotetrasiloxane). Uterine peroxidase activity, a marker for estrogenic activity, was also significantly increased in D4-exposed mice, but not in mice exposed to the other siloxanes. Pretreating mice with the estrogen receptor antagonist ICI 182,780 completely blocked the D4-induced increase in uterine weight, and ovariectomized estrogen receptor-alpha knockout mice showed no increases in uterine weights when orally exposed to D4 or estradiol. In an in vitro estrogen receptor binding assay, D4 showed significant competition with (3)H-estradiol for binding to estrogen receptor-alpha, but not estrogen receptor-beta. The data presented here indicate that D4 has weak estrogenic activity, and that these effects are mediated through estrogen receptor-alpha.

Studies using the estrogen receptor alpha knockout uterus demonstrate that implantation but not decidualization-associated signaling is estrogen dependent.

Ovarian hormonal signaling is essential for proper functioning of the uterus in the establishment of pregnancy. Previous studies have demonstrated that decidualization, a stromal transformation that occurs in response to embryo implantation, can be elicited in the uterus of estrogen receptor alpha knockout (alphaERKO) mice in the absence of the estrogen dependence normally seen in wild-type (WT) mice for this response. While the alphaERKO stromal compartment demonstrated the necessary decidual response, embryo implantation is a process initiated in the epithelial layer, a uterine component that lacks estrogen responsiveness in the alphaERKO. To determine if the alphaERKO uterus would be competent for implantation, donor embryos were transferred into the uterine lumen of WT and alphaERKO females that had been ovariectomized and treated with exogenous estradiol and progesterone to mimic early pregnancy. No implantation occurred in the alphaERKO, while implantation sites containing live embryos were seen in similarly treated WT uteri, indicating that functional estrogen receptor alpha (ERalpha) is required for implantation. Previous observations of estrogen-independent decidualization in the alphaERKO prompted investigation of the mechanism leading to estrogen independence of this process. The disruption of progesterone receptor (PR), Hoxa10, Cox2, or LIF in transgenic mice results in the loss of decidualization response. Therefore, the expression of these genes was studied in WT and alphaERKO uteri by comparing expression following vehicle, progesterone alone (P), or estradiol priming followed by progesterone with nidatory estradiol (E+Pe) and by comparing expression following the above hormonal manipulations in addition to luminal infusion of oil used previously as decidualization-initiating stimulus. The whole-uterus level of PR and Hoxa10 mRNAs did not vary; however, the PR protein was induced in the stroma 24 h after oil infusion. Interestingly, in the WT, this induction was most apparent in samples receiving E+Pe, while in the alphaERKO samples, the induction occurred independent of any hormone priming. Cox2 protein and mRNA increased in both WT and alphaERKO samples 2 h after oil infusion in all three of the treatment groups. In the WT samples, Cox2 levels remained elevated 24 h after oil infusion only in the E+Pe treatment group; however, the elevated Cox2 was seen in samples taken 24 h after oil infusion in all three alphaERKO treatment groups. The alphaERKO uterine tissue appeared to sustain more extensive damage when examined 24 h after oil infusion. Severe trauma, such as crushing of the uterine tissue, has previously been shown to remove the requirement for nidatory estradiol for deciduomas to develop, indicating that the greater susceptibility of alphaERKO uterine tissue to damage from intraluminal oil infusion is contributing to decidualization in the absence of ERalpha. Leukemia inhibitory factor (LIF) mRNA was also induced following estradiol treatment in the WT, but also following oil infusion in WT samples that were not treated with estradiol. In contrast, estradiol does not induce LIF mRNA in the alphaERKO, but oil infusion leads to a robust increase in LIF in all alphaERKO sample groups. LIF binds and activates its membrane receptor, which initiates responses including the phosphorylation and nuclear translocation of Stat3 transcription factor. Thus, Stat3 phosphorylation was studied in WT and alphaERKO samples and found to be induced following oil infusion in all samples. Together, these and previous observations illustrate that estrogen is essential for epithelial proliferation and embryo implantation and that estrogen is dispensable for stromal decidualization in the alphaERKO, as the essential genes and signals required for the response are still induced.

Coumestrol antagonizes neuroendocrine actions of estrogen via the estrogen receptor alpha.

The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor alpha knockout mice (ERalphaKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERalpha. Female wild-type (WT) and ERalphaKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERalphaKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERalphaKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERalpha mediates these effects.

Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice.

Dietary supplements containing concentrates of plant-derived estrogens are being increasingly used by consumers as alternatives for hormone replacement therapy, for treatment of menopausal symptoms, and as cancer preventives. The effect of dietary genistein on dimethylbenz[a]anthracene (DMBA)-induced mammary tumor development was investigated in wild-type (ER alpha WT) and estrogen receptor-alpha knockout (ER alpha KO) mice. ER alpha WT and ER alpha KO mice were fed a casein-based diet containing 0 or 1 g genistein/kg diet from weaning. Tumors were induced by oral administration of DMBA and subscapular implantation of medroxyprogesterone acetate. No tumors were observed in ER alpha KO mice. In ER alpha WT mice, dietary intake of genistein influenced tumor development, enhancing anaplasia of mammary cancer. Mice consuming genistein expressed malignant mammary adenocarcinoma, whereas benign adenomas were observed in mice fed the control diet. Dietary intake was also influenced by genistein, with ER alpha WT and ER alpha KO mice fed genistein consuming less food (p < 0.0001) and subsequently weighing less than mice fed the control diet (p < 0.0001). Significant differences in food intake by genotype were also observed (p = 0.0017), with ER alpha KO mice consuming less than ER alpha WT mice. Overall, this study found no protective effect of genistein on DMBA-induced mammary tumors in mice and suggests a potential adverse effect on tumor development when high levels of genistein are consumed.

Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse.

Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERbeta, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature alphaERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the alphaERKO compared with wild-type. These studies indicate that treatment of alphaERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ERalpha within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the alphaERKO suggests an intraovarian role for ERalpha in follicular development and ovulation.

Retinoid-resistant estrogen receptor-negative human breast carcinoma cells transfected with retinoic acid receptor-alpha acquire sensitivity to growth inhibition by retinoids.

Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each class of these nuclear retinoid receptors is further subdivided into three species, namely alpha, beta, and gamma. Recent studies demonstrate that estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines and tumor samples exhibit significantly higher levels of RAR alpha than their ER-negative counterparts. ER-positive HBC cell lines are sensitive to, and ER-negative cell lines are resistant to, growth inhibitory effects of retinoic acid (RA). We previously demonstrated that the expression of functional ERs in an established ER-negative cell line resulted in higher levels of RAR alpha and sensitivity to growth inhibition by RA. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA in two RA-resistant ER-negative HBC cell lines. Analyses of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Utilizing RAR- and RXR-class selective retinoids, we further demonstrated that only the RAR alpha-selective retinoids mediated the growth inhibition in these cells, while the RXR-selective retinoids were biologically inert. We thus provide evidence that the molecular mechanisms of retinoid inhibition of HBC proliferation predominantly involve RAR alpha.