Comparative Analysis of Metabolites of 'Hongro' Apple Greasiness in Response to Temperature.

Greasiness in apple skin reduces its quality, and its level varies depending on the variety. In this study, low-temperature (1 ± 0.5 °C) stored 'Hongro' and 'Fuji', which had differences in the occurrence of greasiness, were moved to room temperature (20 °C) and untargeted metabolite and fatty acids for skin and flesh along with quality changes due to greasiness occurrence were compared. Ethylene production differed noticeably between the two varieties and increased rapidly in 'Hongro' until 9 d of room-temperature storage. The ethylene production did not differ significantly between the two varieties on day 20 when greasiness occurred. According to the PLS-DA score plot, while 'Hongro' had similar amounts of unsaturated and saturated fatty acids, 'Fuji' had approximately twice as much unsaturated-fatty-acid content. 'Hongro', after 50 d of low-temperature (1 ± 0.5 °C) storage, produced excessive ethylene during room-temperature storage, which was directly related to greasiness development. As a result, the primary wax components of greasy 'Hongro' were nonacosane and nonacosan-10-ol. As the room-temperature storage period elapsed, pentyl linoleate and α-farnesene contents increased significantly. Furthermore, these greasiness-triggering characteristics of 'Hongro' may have been genetically influenced by the paternal parent used during breeding.

Optimization of the ultrasound-assisted extraction of flavonoids and the antioxidant activity of Ruby S apple peel using the response surface method.

A Box-Behnken Design (BBD) was employed to optimize the extraction of antioxidants from Ruby S apple peel by ultrasound-assisted extraction (UAE). The effect of extraction temperature (20-40 °C), extraction time (15-45 min), and ethanol concentration (50-90%) in water on extraction yield, total phenol content (TPC), total flavonoid content (TFC), and DPPH radical scavenging activity of Ruby S peel extracts (RPEs) were investigated. The optimized extraction conditions that maximized extraction yield, TPC, TFC, and DPPH radical scavenging ability, were temperature 20 °C, extraction time 25.30 min, and ethanol concentration 50%. The validity of designed model was verified, and experimental values obtained under optimum conditions concurred with predicted values. Hyperoside, isoquercitrin, and phloridzin, were among the major flavonoids extracted. Our findings demonstrate the suitability of UAE and RSM for the optimization of Ruby S peel extraction and suggest the potential use of RPEs as bioactive functional materials.

Metformin Decreases 2-HG Production through the MYC-PHGDH Pathway in Suppressing Breast Cancer Cell Proliferation.

The biguanide drug metformin has been widely used for the treatment of type 2 diabetes, and there is evidence supporting the anticancer effect of metformin despite some controversy. Here, we report the growth inhibitory activity of metformin in the breast cancer (MCF-7) cells, both in vitro and in vivo, and the associated metabolic changes. In particular, a decrease in a well-known oncometabolite 2-hydroxyglutarate (2-HG) was discovered by a metabolomics approach. The decrease in 2-HG by metformin was accompanied by the reduction in histone methylation, consistent with the known tumorigenic mechanism of 2-HG. The relevance of 2-HG inhibition in breast cancer was also supported by a higher level of 2-HG in human breast cancer tissues. Genetic knockdown of PHGDH identified the PHGDH pathway as the producer of 2-HG in the MCF-7 cells that do not carry isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) mutations, the conventional producer of 2-HG. We also showed that metformin's inhibitory effect on the PHGDH-2HG axis may occur through the regulation of the AMPK-MYC pathway. Overall, our results provide an explanation for the coherent pathway from complex I inhibition to epigenetic changes for metformin's anticancer effect.

Nordihydroguaiaretic Acid as a Novel Substrate and Inhibitor of Catechol O-Methyltransferase Modulates 4-Hydroxyestradiol-Induced Cyto- and Genotoxicity in MCF-7 Cells.

Nordihydroguaiaretic acid (NDGA) is a major lignan metabolite found in Larrea spp., which are widely used in South America to treat various diseases. In breast tissue, estradiol is metabolized to the catechol estrogens such as 4-hydroxyestradiol (4-OHE2), which have been proposed to be cancer initiators potentially involved in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol estrogens to their less toxic methoxy derivatives, such as 4-O-methylestradiol (4-MeOE2). The present study investigated the novel biological activities of NDGA in relation to COMT and the effects of COMT inhibition by NDGA on 4-OHE2-induced cyto- and genotoxicity in MCF-7 human breast cancer cells. Two methoxylated metabolites of NDGA, 3-O-methylNDGA (3-MNDGA) and 4-O-methyl NDGA (4-MNDGA), were identified in the reaction mixture containing human recombinant COMT, NDGA, and cofactors. Km values for the COMT-catalyzed metabolism of NDGA were 2.6 µM and 2.2 µM for 3-MNDGA and 4-MNDGA, respectively. The COMT-catalyzed methylation of 4-OHE2 was inhibited by NDGA at an IC50 of 22.4 µM in a mixed-type mode of inhibition by double reciprocal plot analysis. Molecular docking studies predicted that NDGA would adopt a stable conformation at the COMT active site, mainly owing to the hydrogen bond network. NDGA is likely both a substrate for and an inhibitor of COMT. Comet and apurinic/apyrimidinic site quantitation assays, cell death, and apoptosis in MCF-7 cells showed that NDGA decreased COMT-mediated formation of 4-MeOE2 and increased 4-OHE2-induced DNA damage and cytotoxicity. Thus, NDGA has the potential to reduce COMT activity in mammary tissues and prevent the inactivation of mutagenic estradiol metabolites, thereby increasing catechol estrogen-induced genotoxicities.

Modified Atmosphere and Humidity Film Reduces Browning Susceptibility of Oriental Melon Suture Tissue during Cold Storage.

Oriental melons have a relatively short shelf life as they are harvested during the summer season and susceptible to cold-induced injuries. Typical chilling injury when stored at 4 °C is expressed as browning of the fruit suture. To prolong the shelf life and reduce browning of the fruit, the effects of modified atmosphere packaging (MAP), X-tend modified atmosphere (MA)/modified humidity (MH) bulk packaging (XF), and polyethylene (PE) packaging, on oriental melons were investigated during storage at 4 °C and 10 °C for 14 days and under retail display conditions at 20 °C. The O2 concentrations in PE packages stored at 4 °C and 10 °C ranged from 17.4 to 18.5%, whereas those in XF packages were reduced to 16.3-16.6%. The CO2 content of XF package (4.2-4.6%) was higher than that of PE package (1.4-1.9%) stored at 4 °C or 10 °C. Relative humidity (RH) saturated in the PE packages but not in the XF packages after seven days of storage. Furthermore, PE packages performed better at maintaining melon weight and firmness than XF packages during storage at 10 °C for 14 days and under retail display conditions at 20 °C. PE and XF packages effectively reduced the browning index of the peel and white linear sutures of oriental melons compared with the unpackaged control during cold storage at 4 °C, and this observation was maintained at the retail display condition at 20 °C. The enhanced CO2 levels, reduced O2 levels, and optimal RH values that were provided by the MAP, prevented the browning symptoms, and improved the marketability and shelf life of oriental melons.

Estrogenic properties of Prunus cerasoides extract and its constituents in MCF-7 cell and evaluation in estrogen-deprived rodent models.

Prunus cerasoides (PC) products contain relatively high levels of flavones and isoflavones and may be potential sources of phytoestrogens for postmenopausal symptom relief. We assessed the PC extract (PCE) and its representative constituents in vitro with assays for estrogen receptor alpha binding, estrogen response element transcriptional activity, cell proliferation, and gene expression changes for pS2 in MCF-7 cells. PCE and its compounds showed strong estrogen receptor binding affinities and estrogen response element induction. A previously undescribed compound (designated as compound 18), now identified as being gentisic acid, 5-O-ß-D-(6'-O-trans-4-coumaroyl)-glucopyranoside, also showed potent estrogenic properties and induced proliferation of MCF-7 cells. PCE was evaluated for its in vivo uterotrophic effects in immature female rats as well as for its lipid lowering effects in estrogen-deprived animals. For ovariectomized rats and aged female mice, PCE-treated groups had lower plasma triglyceride levels compared with control and, for the same comparison, had reduced serum levels of liver stress/damage markers. Our results point to strong estrogenic activities and beneficial metabolic effects for PCE, with properties that put PC and its extracts as promising sources of phytoestrogens for symptom relief in menopausal and postmenopausal cases.

Estrogen Receptor-Mediated Transcriptional Activities of Spent Coffee Grounds and Spent Coffee Grounds Compost, and Their Phenolic Acid Constituents.

Spent coffee grounds (SCG) are the most abundant coffee byproduct and are generally discarded as waste. The horticultural use of SCG and SCG compost (SCGC) has become popular due to a growing interest in environmentally friendly measures for waste disposal. Estrogen-like endocrine disrupting chemicals in the soil can be absorbed by plants and subsequently by humans who consume these plants. The objectives of this study are to determine the phytochemical profiles of extracts of SCG and SCGC and to evaluate the estrogen-like activities of SCG, SCGC, and the major coffee phenolic acids, specifically, 5-O-caffeoylquinic acid (CQA), caffeic acid, and ferulic acid. Their inductive effects on estrogen receptor (ER)-mediated gene transcription have been examined in cultured cell lines. CQA was the most abundant phenolic acid in SCG and SCGC and was further examined for its ER-mediated estrogen-like activity using various assays. This is the first study to report the estrogen-like signaling activities of coffee byproducts and their major constituents.

Characterization of Soybean Germinated Embryo Extract as an Estrogen Receptor Subtype-Selective and Tissue-Specific Modulator.

Phytoestrogens possess beneficial effects in the management of menopausal symptoms with few side effects. Soybeans are major natural sources of isoflavones, with high estrogen receptor (ER)-ß selectivity. The objective of this study therefore was to develop a solvent-mediated extraction method for soybean germinated embryos (SGEs) and to investigate the biological activities of the extract. Ethanolic extraction yielded the SGE extract (SGEE), which had a unique composition of biologically active aglycones and soyasaponins. SGEE showed a proliferative effect in MCF7 cells and ERß-selective transcriptional activities in human embryonic kidney cells. In addition, oral administration of SGEE to ovariectomized rats resulted in the induction of ERß and estrogen-responsive genes in the uterus and a decrease in tail skin temperature and uterus weight. Our data suggest that germination and ethanolic extraction are effective measures for producing isoflavone-rich food supplements, which may be useful as alternative menopausal hormone therapy.

Inhibitory and Inductive Effects of Opuntia ficus indica Extract and Its Flavonoid Constituents on Cytochrome P450s and UDP-Glucuronosyltransferases.

Opuntia ficus indica (OFI) is grown abundantly in arid areas and its fruits are regarded as an important food and nutrient source owing to the presence of flavonoids, minerals, and proteins. The previous report that OFI exerts phytoestrogenic activity makes it plausible for OFI-containing supplements to be used as alternative estrogen replacement therapy. In the case of polypharmacy with the consumption of OFI-containing botanicals in post- or peri-menopausal women, it is critical to determine the potential drug-OFI interaction due to the modulation of drug metabolism. In the present study, the modulating effects on the hepatic drug metabolizing enzymes (DMEs) by OFI and its flavonoid constituents (kaempferol, quercetin, isorhamnetin, and their glycosidic forms) were investigated using the liver microsomal fractions prepared from ovariectomized (OVX) rats, human liver microsomes, and human hepatocarcinoma cell line (HepG2). As a result, the oral administration of extracts of OFI (OFIE) in OVX rats induced hepatic CYP2B1, CYP3A1, and UGT2B1. OFIE, hydrolyzed (hdl) OFIE, and several flavonols induced the transcriptional activities of both CYP2B6 and CYP3A4 genes in HepG2 cells. Finally, OFIE did not inhibit activities of cytochrome P450 (CYPs) or uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), whereas hdl OFIE or flavonol treatment inhibited CYP1A2 and CYP3A1/3A4 in rat and human liver microsomes. Our data demonstrate that OFIE may induce or inhibit certain types of DMEs and indicate that drug-OFI interaction may occur when the substrate or inhibitor drugs of specific CYPs or UGTs are taken concomitantly with OFI-containing products.

Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases.

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug⁻herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Estrogenic effects of phytoestrogens derived from Flemingia strobilifera in MCF-7 cells and immature rats.

Phytoestrogen (PE) has received considerable attention due to the physiological significance of its estrogenicity. Flemingia strobilifera (FS) has been used as a folk medicine in Asia for the treatment of inflammation, cancer, and infection; however, the estrogenic effects and chemical components of FS have not yet been reported. We aimed to uncover the estrogenic properties and PEs derived from FS using phytochemical and pharmacological evaluation. PEs from FS extract (FSE) were analyzed by NMR, HPLC, and MS. To evaluate estrogenic activity, FSE and its compounds were evaluated by in vitro and in vivo assays, including human estrogen receptor alpha (hERα) binding, estrogen response element (ERE)-luciferase reporter assays, and uterotrophic assays. FSE and its compounds 1-5 showed binding affinities for hERα and activated ERE transcription in MCF-7 cells. Additionally, FSE and compounds 1-5 induced MCF-7 cell proliferation and trefoil factor 1 (pS2) expression. In immature female rats, significant increases in uterine weight and pS2 gene were observed in FSE-treated groups. We identified estrogenic activities of FSE and its bioactive compounds, suggesting their possible roles as PEs via ERs. PEs derived from FSE are promising candidates for ER-targeted therapy for post-menopausal symptoms.

Spiroketones and a Biphenyl Analog from Stems and Leaves of Larrea nitida and Their Inhibitory Activity against IL-6 Production.

Bioactivity-guided fractionation for the stems of leaves of Larrea nitida Cav., using interleukin-6 (IL-6) inhibitory assay in human mast cells (HMC-1), led to the isolation of three new compounds with an unprecedented skeleton in nature (1-3) and three known compounds (4-6). Their structures were elucidated through extensive spectroscopic analysis. The three new compounds were elucidated as two new spiroketones, nitidaones A (1), and B (2) and one new biphenyl analog, nitidaol (3). The known compounds were identified as nordihydroguaiaretic acid (4), 7,3',4'-tri-O-methylquercetin (5) and ayanin (6). All the isolates were tested for their inhibitory activity against IL-6 production in HMC-1 cells. Of them, compounds 1, 3-6 showed potent anti-inflammatory activity, with IC50 values of 12.8, 17.5, 14.9, 22.9, and 17.8 µM, respectively.

Characterization of Phase I and Phase II Hepatic Metabolism and Reactive Intermediates of Larrea nitida Cav. and Its Lignan Compounds.

Larrea nitida Cav. (LNC), which belongs to the family Zygophyllaceae, is widely indigenous and used in South America to treat various pathological conditions. It contains the antioxidant and antiinflammatory but toxic nordihydroguaiaretic acid (NDGA) as well as O-methylated metabolite of NDGA (MNDGA) as bioactive compounds. The hepatic metabolism-based toxicological potential of extracts of LNC (LNE), NDGA, and MNDGA has not previously been reported. The present study aimed to characterize the phase I and phase II hepatic metabolism and reactive intermediates of LNE, NDGA, and MNDGA and their effects on the major drug-metabolizing enzymes in vitro and ex vivo. A methanol extract of LNC collected from Chile as well as NDGA and MNDGA isolated from LNE were subjected to metabolic stability assays in liver microsomes in the presence of the cofactors reduced nicotinamide dinucleotide phosphate (NADPH) and/or uridine 5'-diphosphoglucuronic acid (UDPGA). Cytochrome P450 (CYP) inhibition assays were performed using CYP isozyme-specific model substrates to examine the inhibitory activities of LNE, NDGA, and MNDGA, which were expressed as % inhibition and IC50 values. Ex vivo CYP induction potential was investigated in the liver microsomes prepared from the rats intraperitoneally administered with LNE. Glutathione (GSH) adduct formation was monitored by LC-MS3 analysis of the microsomal incubation samples with either NDGA or MNDGA and an excess of GSH to determine the formation of electrophilic reactive intermediates. Both NDGA and MNDGA were stable to NADPH-dependent phase I metabolism, but labile to glucuronide conjugation. LNE, NDGA, and MNDGA showed significant inhibitory effects on CYP1A2, 2C9, 2D6, and/or 3A4, with IC50 values in the micromolar range. LNE was found to be a CYP1A2 inducer in ex vivo rat experiments, and mono- and di-GSH adducts of both NDGA and MNDGA were identified by LC-MS3 analysis. Our study suggests that hepatic clearance is the major elimination route for the lignans NDGA and MNDGA present in LNE. These lignans may possess the ability to modify biomacromolecules via producing reactive intermediates. In addition, LNE, NDGA, and MNDGA are found to be inhibitors for various CYP isozymes such as CYP2C9 and 3A4. Thus, the consumption of LNC as an herbal preparation or NDGA may cause metabolism-driven herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of the Biological Activity of Opuntia ficus indica as a Tissue- and Estrogen Receptor Subtype-Selective Modulator.

Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17ß-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

Metformin inhibits early stage diethylnitrosamine­induced hepatocarcinogenesis in rats.

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)­induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma­specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S­transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK­dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high­risk patients, including chronic hepatitis and cirrhosis.

Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells.

Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 µM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

Inhibition of Aerobic Glycolysis Represses Akt/mTOR/HIF-1α Axis and Restores Tamoxifen Sensitivity in Antiestrogen-Resistant Breast Cancer Cells.

Tamoxifen resistance is often observed in the majority of estrogen receptor-positive breast cancers and it remains as a serious clinical problem in breast cancer management. Increased aerobic glycolysis has been proposed as one of the mechanisms for acquired resistance to chemotherapeutic agents in breast cancer cells such as adriamycin. Herein, we report that the glycolysis rates in LCC2 and LCC9--tamoxifen-resistant human breast cancer cell lines derived from MCF7--are higher than those in MCF7S, which is the parent MCF7 subline. Inhibition of key glycolytic enzyme such as hexokinase-2 resulted in cell growth retardation at higher degree in LCC2 and LCC9 than that in MCF7S. This implies that increased aerobic glycolysis even under O2-rich conditions, a phenomenon known as the Warburg effect, is closely associated with tamoxifen resistance. We found that HIF-1α is activated via an Akt/mTOR signaling pathway in LCC2 and LCC9 cells without hypoxic condition. Importantly, specific inhibition of hexokinase-2 suppressed the activity of Akt/mTOR/HIF-1α axis in LCC2 and LCC9 cells. In addition, the phosphorylated AMPK which is a negative regulator of mTOR was decreased in LCC2 and LCC9 cells compared to MCF7S. Interestingly, either the inhibition of mTOR activity or increase in AMPK activity induced a reduction in lactate accumulation and cell survival in the LCC2 and LCC9 cells. Taken together, our data provide evidence that development of tamoxifen resistance may be driven by HIF-1α hyperactivation via modulation of Akt/mTOR and/or AMPK signaling pathways. Therefore, we suggest that the HIF-1α hyperactivation is a critical marker of increased aerobic glycolysis in accordance with tamoxifen resistance and thus restoration of aerobic glycolysis may be novel therapeutic target for treatment of tamoxifen-resistant breast cancer.

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator.

Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

Selective Estrogen Receptor Modulation by Larrea nitida on MCF-7 Cell Proliferation and Immature Rat Uterus.

Larrea nitida is a plant that belongs to the Zygophyllaceae family and is widely used in South America to treat inflammatory diseases, tumors and menstrual pain. However, its pharmacological activity remains unclear. In this study we evaluated the property of selective estrogen receptor modulator (SERM) of Larrea nitida extracts (LNE) as a phytoestrogen that can mimic, modulate or disrupt the actions of endogenous estrogens, depending on the tissue and relative amount of other SERMs. To investigate the property of SERM of LNE, we performed MCF-7 cell proliferation assays, estrogen response element (ERE)-luciferase reporter gene assay, human estrogen receptor (hER) binding assays and in vivo uterotrophic assay. To gain insight into the active principles, we performed a bioassay-guided analysis of LNE employing solvents of various polarities and using classical column chromatography, which yielded 16 fractions (LNs). LNE showed high binding affinities for hERα and hERß with IC50 values of 1.20 ×10(-7) g/ml and 1.00×10(-7) g/ml, respectively. LNE induced 17ß-estradiol (E2)-induced MCF-7 cell proliferation, however, it reduced the proliferation in the presence of E2. Furthermore, LNE had an atrophic effect in the uterus of immature rats through reducing the expression level of progesterone receptor (PR) proteins. LN08 and LN10 had more potent affinities for binding on hER α and ß than other fractions. Our results indicate that LNE had higher binding affinities for hERß than hERα, and showed SERM properties in MCF-7 breast cancer cells and the rat uterus. LNE may be useful for the treatment of estrogen-related conditions, such as female cancers and menopause.