Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer-Dox-AGM in breast cancer cells

Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity. (c) 2006 Elsevier B.V All rights reserved.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells

Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10-4 M methylparaben, 10–5 M n-propylparaben or 10–5 M n-butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose-responses showed that concentrations as low as 10–6 M methylparaben, 10–7 M n-propylparaben and 10–7 M n-butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

Effect of aluminium on migration of oestrogen unresponsive MDA-MB-231 human breast cancer cells in culture

Aluminium (Al) has been measured in human breast tissue, and may be a contributory factor in breast cancer development. At the 10th Keele meeting, we reported that long-term exposure to Al could increase migratory properties of oestrogen-responsive MCF-7 human breast cancer cells suggesting a role for Al in the metastatic process. We now report that long-term exposure (20–25 weeks) to Al chloride or Al chlorohydrate at 10−4 M or 10−5Mconcentrations can also increase themigration of oestrogen unresponsiveMDA-MB-231 human breast cancer cells as measured using time-lapse microscopy and xCELLigence technology. In parallel, Al exposure was found to give rise to increased secretion of active matrixmetalloproteinaseMMP9 as measured by zymography, and increased intracellular levels of activated MMP14 as measured by western immunoblotting. These results demonstrate that Al can increase migration of human breast cancer cells irrespective of their oestrogen responsiveness, and implicate alterations to MMPs as a potential mechanism worthy of further study.

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268±28 g/l) compared with control healthy subjects (mean 131±10 g/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150g/l) compared with human serum (median 6g/l) or human milk (median 25g/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.

Exploring quercetin and luteolin derivatives as antiangiogenic agents

The formation of new blood vessels from the pre-existing vasculature (angiogenesis) is a crucial stage in cancer progression and, indeed, angiogenesis inhibitors are now used as anticancer agents, clinically. Here we have explored the potential of flavonoid derivatives as antiangiogenic agents. Specifically, we have synthesised methoxy and 4-thio derivatives of the natural flavones quercetin and luteolin, two of which (4-thio quercetin and 4-thio luteolin) had never been previously reported. Seven of these compounds showed significant (P<0.05) antiangiogenic activity in an in vitro scratch assay. Their activity ranged from an 86% inhibition of the vascular endothelium growth factor (VEGF)-stimulated migration (observed for methoxyquercetin at 10 µM and for luteolin at 1 µM) to a 36% inhibition (for thiomethoxy quercetin at 10 µM). Western blotting studies showed that most (4 out of 7) compounds inhibited phosphorylation of the VEGF receptor-2 (VEGFR2), suggesting that the antiangiogenic activity was due to an interference with the VEGF/VEGFR2 pathway. Molecular modelling studies looking at the affinity of our compounds towards VEGFR and/or VEGF confirmed this hypothesis, and indeed the compound with the highest antiangiogenic activity (methoxyquercetin) showed the highest affinity towards VEGFR and VEGF. As reports from others have suggested that structurally similar compounds can elicit biological responses via a non-specific, promiscuous membrane perturbation, potential interactions of the active compounds with a model lipid bilayer were assessed via DSC. Luteolin and its derivatives did not perturb the model membrane even at concentrations 10 times higher than the biologically active concentration and only subtle interactions were observed for quercetin and its derivatives. Finally, cytotoxicity assessment of these flavonoid derivatives against MCF-7 breast cancer cells demonstrated also a direct anticancer activity albeit at generally higher concentrations than those required for an antiangiogenic effect (10 fold higher for the methoxy analogues). Taken together these results show promise for flavonoid derivatives as antiangiogenic agents.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Antagonistic potential of bacterial isolates associated with entomopathogenic nematodes against tomato wilt caused by Fusarium oxysporum f.sp., Lycopersici under greenhouse conditions

Three concentrations of Xenorhabdus nematophila and Xenorhabdus spp., (4x10(5,) 4x10(6,) 4x10(7) cells/ml) were evaluated in the laboratory and in pot experiments to test their antagonistic effects on Fusarium oxysporum f.sp., lycopersici. All concentrations effectively inhibited its growth on agar plates. In soil under greenhouse conditions treatments with each bacterium at 4x10(7) cells/ml reduced the disease incidence of tomato by up to 40.38 and 47.54% respectively and there were significant increases of plant biomass by 198 and 211% respectively. The rhizosphere population of Fusarium oxysporum f.sp., lycopersici was reduced by 97%. The Xenorhabdus spp., was comparatively more effective than X. nematophila.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 1010.7 cells/g feces; antibiotic-treated, 108.4). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (109.1 cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.

Polysialic acid as a drug carrier: evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers

This paper explores the potential of polysialic acid (PSA) as a carrier for low molecular weight anticancer drugs. A PSA–epirubicin (Epi) conjugate was synthesized and compared against Epi conjugates containing established carriers, namely: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, poly(ethylene glycol) (PEG) and polyglutamic acid (PGA). Biological assessments in the breast cancer cell line MCF-7 and in the anthracycline resistant MCF-7/DX showed that the PSA–Epi conjugate had the highest activity (40% and 30% cell death in the two cell lines at 1 mM Epi equiv., respectively). FACS studies confirmed internalization of all conjugates by cholesterol-dependent endocytosis. PSA–Epi showed release of Epi (40% at 5 h) when incubated with lysosome extracts. In vivo evaluation showed that all conjugates had a significantly longer half-life compared to free Epi. This study also allowed an investigation on the effect of the polymeric carrier on the biological activity of a conjugate, with the biodegradability of the carrier emerging as an important feature.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

Novel synthesised flavone derivatives provide significant insight into the structural features required for enhanced anti-proliferative activity

With many cancers showing resistance to current chemotherapies, the search for novel anti-cancer agents is attracting considerable attention. Natural flavonoids have been identified as useful leads in such programmes. However, since an in-depth understanding of the structural requirements for optimum activity is generally lacking, further research is required before the full potential of flavonoids as anti-proliferative agents can be realised. Herein a broad library of 76 methoxy and hydroxy flavones, and their 4-thio analogues, was constructed and their structure-activity relationships for anti-proliferative activity against the breast cancer cell lines MCF-7 (ER+ve), MCF-7/DX (ER+ve, anthracycline resistant) and MDA-MB-231 (ER-ve) were probed. Within this library, 42 compounds were novel, and all compounds were afforded in good yields and > 95% purity. The most promising lead compounds, specifically the novel hydroxy 4-thioflavones 15f and 16f, were further evaluated for their anti-proliferative activities against a broader range of cancer cell lines by the National Cancer Institute (NCI), USA and displayed significant growth inhibition profiles (e.g Compound-15f: MCF-7 (GI50 = 0.18 μM), T-47D (GI50 = 0.03 μM) and MDA-MB-468 (GI50 = 0.47 μM) and compound-16f: MCF-7 (GI50 = 1.46 μM), T-47D (GI50 = 1.27 μM) and MDA-MB-231 (GI50 = 1.81 μM). Overall, 15f and 16f exhibited 7-46 fold greater anti-proliferative potency than the natural flavone chrysin (2d). A systematic structure-activity relationship study against the breast cancer cell lines highlighted that free hydroxyl groups and the B-ring phenyl groups were essential for enhanced anti-proliferative activities. Substitution of the 4-C=O functionality with a 4-C=S functionality, and incorporation of electron withdrawing groups at C4’ of the B-ring phenyl, also enhanced activity. Molecular docking and mechanistic studies suggest that the anti-proliferative effects of flavones 15f and 16f are mediated via ER-independent cleavage of PARP and downregulation of GSK-3β for MCF-7 and MCF-7/DX cell lines. For the MDA-MB-231 cell line, restoration of the wild-type p53 DNA binding activity of mutant p53 tumour suppressor gene was indicated.

The intracellular genistein metabolite 5,7,3 ',4 '-tetrahydroxyisoflavone mediates G2-M cell cycle arrest in cancer cells via modulation of the p38 signaling pathway

The cellular actions of genistein are believed to mediate the decreased risk of breast cancer associated with high soy consumption. We have investigated the intracellular metabolism of genistein in T47D tumorigenic and MCF-10A nontumorigenic cells and assessed the cellular actions of resultant metabolites. Genistein selectively induced growth arrest and G2-M phase cell cycle block in T47D but not MCF10A breast epithelial cells. These antiproliferative effects were paralleled by significant differences in the association of genistein to cells and in particular its intracellular metabolism. Genistein was selectively taken up into T47D cells and was subject to metabolism by CYP450 enzymes leading to the formation of both 5,7,3',4'-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF THIF inhibited cdc2 activation via the phosphorylation of p38 MAP kinase, suggesting that this species may mediate genistein's cellular actions. THIF exposure activated p38 and caused subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of the cdc2-cyclin B1 complex. We suggest that the formation of THIF may mediate the cellular actions of genistein in tumorigenic breast epithelial cells via the activation of signaling through p38. (c) 2006 Elsevier Inc. All rights reserved.