Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

The original publication is available at http://pubs.acs.org/Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERβ but not via ERα. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERβ. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species. © 2007 American Chemical Society.Publishers' versio

Disease- and treatment-associated acquired glucocorticoid resistance

The development of resistance to glucocorticoids (GCs) in therapeutic regimens poses a major threat. Generally, GC resistance is congenital or acquired over time as a result of disease progression, prolonged GC treatment or, in some cases, both. Essentially, disruptions in the function and/or pool of the glucocorticoid receptor α (GRα) underlie this resistance. Many studies have detailed how alterations in GRα function lead to diminished GC sensitivity; however, the current review highlights the wealth of data concerning reductions in the GRα pool, mediated by disease-associated and treatment-associated effects, which contribute to a significant decrease in GC sensitivity. Additionally, the current understanding of the molecular mechanisms involved in driving reductions in the GRα pool is discussed. After highlighting the importance of maintaining the level of the GRα pool to combat GC resistance, we present current strategies and argue that future strategies to prevent GC resistance should involve biased ligands with a predisposition for reduced GR dimerization, a strategy originally proposed as the SEMOGRAM–SEDIGRAM concept to reduce the side-effect profile of GCs

The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor

Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ∼24 nM for transactivation of the anti-inflammatory GILZ gene and ∼4-20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells

Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

The original publication is available at http://pubs.acs.org/Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERβ but not via ERα. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERβ. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species. © 2007 American Chemical Society.Publishers' versio

Acquired Glucocorticoid Resistance Due to Homologous Glucocorticoid Receptor Downregulation: A Modern Look at an Age-Old Problem

For over 70 years, the unique anti-inflammatory properties of glucocorticoids (GCs), which mediate their effects via the ligand-activated transcription factor, the glucocorticoid receptor alpha (GRα), have allowed for the use of these steroid hormones in the treatment of various autoimmune and inflammatory-linked diseases. However, aside from the onset of severe side-effects, chronic GC therapy often leads to the ligand-mediated downregulation of the GRα which, in turn, leads to a decrease in GC sensitivity, and effectively, the development of acquired GC resistance. Although the ligand-mediated downregulation of GRα is well documented, the precise factors which influence this process are not well understood and, thus, the development of an acquired GC resistance presents an ever-increasing challenge to the pharmaceutical industry. Recently, however, studies have correlated the dimerization status of the GRα with its ligand-mediated downregulation. Therefore, the current review will be discussing the major role-players in the homologous downregulation of the GRα pool, with a specific focus on previously reported GC-mediated reductions in GRα mRNA and protein levels, the molecular mechanisms through which the GRα functional pool is maintained and the possible impact of receptor conformation on GC-mediated GRα downregulation

DataSheet1_Combinatorial treatments of tamoxifen and SM6Met, an extract from Cyclopia subternata Vogel, are superior to either treatment alone in MCF-7 cells.docx

Synergistic drug combinations are not only popular in antibiotic, anti-microbial, immune disease (i.e., AIDS) and viral infection studies, but has also gained traction in the field of cancer research as a multi-targeted approach. It has the potential to lower the doses needed of standard of care (SOC) therapeutic agents, whilst maintaining an effective therapeutic level. Lower dosages could ameliorate the fundamental problems such as drug resistance and metastasis associated with current SOC therapies. In the current study, we show that the combination of SM6Met with (2)-4-hydroxytamoxifen (4-OH-Tam, the active metabolite of tamoxifen) produces a strong synergistic effect in terms of inhibiting MCF7 ER-positive (ER+) breast cancer cell proliferation and that a 20 times lower dose of 4-OH-Tam in combination with SM6Met is required to produce the same inhibitory effect on cell proliferation as 4-OH-Tam on its own. Cell cycle analyses of the best combination ratios of SM6Met and 4-OH-Tam also suggests that the combination results in increased accumulation of cells in the S-phase and in the apoptotic phase. Moreover, the best combination ratio (20:1) of SM6Met with 4-OH-Tam displayed greater anti-metastatic potential in terms of inhibiting ER+ breast cancer cell migration, invasion, and colony formation than the SOC therapy alone, suggesting that SM6Met together with 4-OH-Tam could be a viable drug combination for not only delaying resistance and ameliorating the negative side-effects associated with current SOC therapies, like tamoxifen, but could also provide a novel, more affordable therapeutic alternative for treating or preventing ER+ breast cancer metastasis.</p

Only GR protein is detected in primary cervical epithelial cells (VEN-100).

<p>(<b>A</b>) Upon arrival the VEN-100 cells were rested overnight before being washed once with PBS and harvested with TRIzol®. Total RNA was isolated and 500 ng RNA was reverse-transcribed. Steroid receptor gene expression was measured by qRT-PCR with receptor-specific primers, followed by gel electrophoresis to confirm the PCR products. (<b>B</b>) VEN-100 cells were rested overnight before harvesting in 2X SDS sample buffer. COS-1 cells were transiently transfected with 1 µg/well pcDNA3 (empty vector) which served as negative control (−CTRL) or with 1 µg/well steroid receptor expression vectors (GR, PR-B, AR, MR and ERα) which served as positive controls (+CTRL). Twenty fourhrs later, the COS-1 cells were washed once and lysed with 2X SDS sample buffer. Equal volumes of cell lysate (VEN-100 and COS-1 ctrls) were analysed by Western blotting with antibodies specific for the GR and Flotillin-1 (loading control), respectively.</p

The GR at least in part directly regulates mRNA levels of the inflammatory genes.

<p>End1/E6E7 cells were pretreated with 1 µg/ml cycloheximide (CHX) then treated for 24 hrs with 100 nM DEX, MPA, P4, NET-A or vehicle (ethanol) (CTRL), in the absence or presence of CHX. Total RNA was isolated and reverse-transcribed. Relative (<b>A</b>) GILZ (<b>B</b>) IκBα, (<b>C</b>) RANTES, (<b>D</b>) IL-6 and (<b>E</b>) IL-8 gene expressions was measured by real-time qRT-PCR and normalised to GAPDH mRNA expression. In addition, relative gene expressions were normalized to basal activity (CTRL) in order to obtain relative fold expression. Graphs represent pooled results of at least three independent experiments and are plotted as mean ± SEM. To verify that the CHX inhibited <i>de novo</i> protein synthesis, End1/E6E7 cells were pretreated with CHX then treated with 100 nM DEX or vehicle (ethanol) (CTRL) for 24 hrs. (<b>F</b>) Cells were harvested and equal volumes of lysate were analysed by Western blotting with an antibody specific for IκBα and a GAPDH specific antibody as loading control. (<b>G</b>) Western blots of four independent experiments were quantified to determine the relative GR protein expression. Statistical analysis was carried out using GraphPad Prism software (version 5) using a one-way ANOVA with a Dunnett post-test followed by a student’s t-test to compare specific conditions to each other. Statistical significance is denoted by *, ** or *** to indicate P<0.05, P<0.001 or P<0.0001, respectively.</p