Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE: Progesterone (P(4)) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri. METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro. RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors. CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation

Growth Hormone potentiates 17beta-estradiol-dependent breast cancer cell proliferation in an IGF-I-independent manner

Estrogen action in mammary gland development and breast cancer progression is tightly linked to the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis. In rodents, both estradiol (E2) and GH are necessary for gland development and carcinogenesis. However, to what extent the effects of GH are mediated by IGF-I is unclear. Here, we demonstrate in Spontaneous Dwarf rats, which lack endogenous GH, that both GH and estradiol (E2) are critical to maintain proliferation of normal and cancerous mammary epithelial cells. In T47D human breast cancer cells, GH significantly enhances E2-stimulated proliferation. While the in vivo effects of GH may be direct on mammary epithelial cells and/or mediated by increased IGF-I, GH action in T47D cells was independent of IGF-I expression and IGF-IR signaling, suggesting that GH also may exert direct effects on breast cancer cells. Use of an IGF-IR inhibitor demonstrated that while E2-dependent proliferation required IGF-IR signaling, the combination of GH+E2 overcame IGF-IR blockade, restoring proliferation. In contrast, studies with specific inhibitors indicate that GH action through both Jak2 and EGFR was required for subsequent ERK activation and was essential for potentiation of E2-dependent proliferation. Downstream of these pathways, we identified a number of immediate early response genes associated with proliferation that are rapidly and robustly up-regulated by GH. Taken together, these findings demonstrate that GH can have important effects in breast cancer cells that are distinct from IGF-I, suggesting that novel drugs or improved combination therapies targeting ER and the GH/IGF axis may be beneficial for breast cancer patients

Structural Modulation of Oxidative Metabolism in Design of Improved Benzothiophene Selective Estrogen Receptor Modulators

Raloxifene and arzoxifene are benzothiophene selective estrogen receptor modulators (SERMs) of clinical use in postmenopausal osteoporosis and treatment of breast cancer and potentially in hormone replacement therapy. The benefits of arzoxifene are attributed to improved bioavailability over raloxifene, whereas the arzoxifene metabolite, desmethylarzoxifene (DMA) is a more potent antiestrogen. As polyaromatic phenolics, benzothiophene SERMs undergo oxidative metabolism to electrophilic quinoids. The long-term clinical use of SERMs demands increased understanding of correlations between structure and toxicity, with metabolism being a key component. A homologous series of 4′-substituted 4′-desmethoxyarzoxifene derivatives was developed, and metabolism was studied in liver and intestinal microsomes. Formation of glutathione conjugates was assayed in rat liver microsomes and novel adducts were characterized by liquid chromatography-tandem mass spectrometry. Formation of glucuronide conjugates was assayed in human intestine and liver microsomes, demonstrating formation of glucuronides ranging from 5 to 100% for the benzothiophene SERMs: this trend was inversely correlated with the loss of parent SERM in rat liver microsomal incubations. Molecular orbital calculations generated thermodynamic parameters for oxidation that correlated with Hammett substituent constants; however, metabolism in liver microsomes correlated with a combination of both Hammett and Hansch lipophilicity parameters. The results demonstrate a rich oxidative chemistry for the benzothiophene SERMs, the amplitude of which can be powerfully modulated, in a predictable manner, by structural tuning of the 4′-substituent. The predicted extensive metabolism of DMA was confirmed in vivo and compared with the relatively stable arzoxifene and F-DMA

Breast cancer prevention with liquiritigenin from licorice through the inhibition of aromatase and protein biosynthesis in high-risk women’s breast tissue

Abstract Breast cancer risk continues to increase post menopause. Anti-estrogen therapies are available to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer prevention. Natural products such as hops (Humulus lupulus) and three pharmacopeial licorice (Glycyrrhiza) species have demonstrated estrogenic and chemopreventive properties, but little is known regarding their effects on aromatase expression and activity as well as pro-proliferation pathways in human breast tissue. We show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plant extracts. Moreover, phytoestrogens such as liquiritigenin which is common in all licorice species have potent aromatase inhibitory activity, which is further supported by computational docking of their structures in the binding pocket of aromatase. In addition, GI extract and liquiritigenin suppress aromatase expression in the breast tissue of high-risk postmenopausal women. Although liquiritigenin has estrogenic effects in vitro, with preferential activity through estrogen receptor (ER)-β, it reduces estradiol-induced uterine growth in vivo. It downregulates RNA translation, protein biosynthesis, and metabolism in high-risk women’s breast tissue. Finally, it reduces the rate of MCF-7 cell proliferation, with repeated dosing. Collectively, these data suggest that liquiritigenin has breast cancer prevention potential for high-risk postmenopausal women

Bioactivity-Guided Isolation of Totarane-Derived Diterpenes from Podocarpus neriifolius and Structure Revision of 3-Deoxy-2α-hydroxynagilactone E

Abstract Bioactivity-guided phytochemical investigation of Podocarpus neriifolius D. Don. (Podocarpaceae) has led to the isolation of one new (2) and three known (1, 3, and 4) B-type podolactones, along with three totarane-type diterpenes (5-7). Their structures were determined by interpretation of High Resolution ElectroSpray Ionization Mass Spectrometry (HRESIMS) and 1D and 2D NMR data, and comparison with the values reported in the literature. The structure of compound 1, previously identified as 3-deoxy-2α-hydroxynagilactone E (8), was revised as its 2β-epimer, which has been reported recently as a new compound. All of the isolates were evaluated for their antiproliferative activity against a panel of four human cancer cell lines, namely, ovarian (OVCAR3), breast (MDA-MB-231), colon (HT-29), and melanoma (MDA-MB-435), and compounds 1 and 3 were found to be cytotoxic with IC50 values in the low micromolar range for most of the cell lines used. The major compound, inumakilactone A (3), was further tested in vivo using the HT-29, MDA-MB-435, and OVCAR3 cells in a murine hollow fiber model, for the first time. Graphical Abstrac

Induction of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) by <i>Glycyrrhiza</i> Species Used for Women’s Health: Differential Effects of the Michael Acceptors Isoliquiritigenin and Licochalcone A

For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme, NAD­(P)­H:quinone oxidoreductase 1 (NQO1), <i>in vitro</i> and <i>in vivo</i>. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in <i>Glycyrrhiza glabra</i> (GG), <i>G. uralensis</i> (GU), <i>G. inflata</i> (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human hepatoma (HepG2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in nontumorigenic breast epithelial MCF-10A cells. In female Sprague–Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 <i>in vivo</i> most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability <i>in vivo</i>. These data show the chemopreventive potential of licorice species <i>in vitro</i> could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the <i>in vivo</i> effects in the rat model after four-day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo

Abstract Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms

Growth Hormone Potentiates 17β-Estradiol-Dependent Breast Cancer Cell Proliferation Independently of IGF-I Receptor Signaling

Estrogen action in mammary gland development and breast cancer progression is tightly linked to the GH/IGF-I axis. Although many of the effects of GH on mammary gland growth and development require IGF-I, the extent to which GH action in breast cancer depends on IGF-I is not known. We examined GH action in a panel of estrogen receptor-positive breast cancer cell lines and found that T47D cells express significant levels of GH receptor and that GH significantly enhances 17β-estradiol (E2)-stimulated proliferation in these cells. GH action in the T47D cells was independent of changes in IGF-I and IGF-I receptor (IGF-IR) expression and IGF-IR signaling, suggesting that GH can exert direct effects on breast cancer cells. Although E2-dependent proliferation required IGF-IR signaling, the combination of GH+E2 overcame inhibition of IGF-IR activity to restore proliferation. In contrast, GH required both Janus kinase 2 and epidermal growth factor receptor signaling for subsequent ERK activation and potentiation of E2-dependent proliferation. Downstream of these pathways, we identified a number of immediate early-response genes associated with proliferation that are rapidly and robustly up-regulated by GH. These findings demonstrate that GH can have important effects in breast cancer cells that are distinct from IGF-IR activity, suggesting that novel drugs or improved combination therapies targeting estrogen receptor and the GH/IGF axis may be beneficial for breast cancer patients