Destabilizing domains derived from the human estrogen receptor

Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.     

A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.     

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

Background  

Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF₁₆₅) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro.     

Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Background

Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment.

Results

The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface.

Conclusion

We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.     

Aspirin-induced Bcl-2 translocation and its phosphorylation in the nucleus trigger apoptosis in breast cancer cells

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.     

Electrochemical assay of the relationship between the inhibition of phosphatidylinositol 3-kinase pathway and estrogen receptor expression in breast cancer

Breast cancer has become one of the most threatening diseases to women throughout the world. Emerging evidence implies that estrogen receptor (ER) and phosphatidylinositol 3-kinase (PI3K) pathways play central roles in both breast cancer progression and response to therapy. In this work, we have probed into ER expression related to the PI3K pathway at the protein level with an electrochemical technique based on the detection of ER proteins in nuclear extracts with an Exonuclease III protection-based strategy. Experimental results show that an increased number of ER proteins can be detected upon PI3K inhibition, demonstrating the reversal effect of the PI3K inhibitor on ER expression. Moreover, treatment with different concentrations of the PI3K inhibitor NVP-BEZ235 can result in a dose-dependent alteration of ER protein levels, implying an intimate link between ER and PI3K pathways. This work may be a great help to understand the mysteries underlying PI3K-related endocrine resistance and to evaluate the effect of therapeutic interventions in the future.     

Solid-phase synthesis of tetrasubstituted pyrazoles, novel ligands for the estrogen receptor

Most ligands for the estrogen receptor (ER) are not well suited for synthesis by combinatorial means, because their construction involves a series of carbon-carbon bond forming reactions that are not uniformly high yielding. In previous work directed to overcoming this limitation, we surveyed various phenol-substituted five-membered heterocycles, hoping to find a system that would afford both high ER binding affinity and whose synthesis could be adapted to solid-phase methods (Fink et al. Chem. Biol. 1999, 6, 206-219.) In this report, we have developed a reliable and efficient solid-phase method to prepare the best of these heterocycles, the tetrasubstituted pyrazoles, and we have used this methodology to produce small, discrete libraries of these novel ER ligands. We used a combination of FT-IR and nanoprobe (1)H NMR-MAS to characterize intermediates leading up to the final pyrazole products directly on the bead. We also developed a scavenging resin, which enabled us to obtain products free from inorganic contaminants. We prepared a 12-member test library, and then a 96-member library, and in both cases we determined product purity and ER binding affinity of all of the library members. Several interesting binding affinity patterns have emerged from these studies, and they have provided us with new directions for further exploration, which has led to pyrazoles having high affinity and potency as agonists and antagonists toward the ER alpha subtype.     

Valtrate from Valeriana jatamansi Jones induces apoptosis and inhibits migration of human breast cancer cells in vitro

Abstract

Valtrate is a principle compound isolated from Valeriana jatamansi Jones, a traditional Chinese folk medicine originally used to treat various nervous disorders. Here, we found that valtrate exhibited significant anti-cancer activity in vitro, especially in human breast cancer cells, while displayed relatively low cytotoxicity to normal human breast epithelial cells (MCF 10A). Valtrate induced cell cycle arrest at G2/M stage and apoptosis in MDA-MB-231 and MCF-7 cells, with reduced expression of p-Akt (Ser 473), cyclin B1 and caspase 8, and increased expression of p21, p-cdc2, cleaved-caspase 3, cleaved-caspase 7 and poly (ADP-ribose) polymerase (PARP). In addition, valtrate inhibited cell migration through down-regulation of MMP-9 and MMP-2 expression. These results demonstrate that valtrate possesses anti-breast cancer activities via cell cycle arrest, apoptosis, and inhibition of cell migration, thus supporting valtrate as a potential antitumor agent.     

Homology modeling of the receptor binding domain of human thrombopoietin

Platelet production in blood is regulated by a lineage specific humoral factor, thrombopoietin (TPO). The amino terminal domain of TPO (TPO-N) is responsible for the signal transduction mediated by the TPO receptor, c-mpl. From the predicted length of helices we found that TPO-N belongs to the long-chain subfamily of the four-helix bundle cytokine family. We built a three dimensional model of TPO-N by a comparative homology modeling procedure. The four helices of TPO-N with an up-up-down-down topology are stabilized by a tightly packed central hydrophobic core and the extended loop AB makes an additional hydrophobic core with helices B and D outside of the four helix bundle scaffold. An interpretation of the previous site directed mutageneses results in light of the model enabled us to identify two isolated receptor binding sites. The surface made of Lys 136, Lys 138 and Lys 140 in helix D, and Pro 42 and Glu 50 in loop AB forms the first receptor binding site, while the surface of Asp 8, Arg 10 and Lys14 in helix A represents the second binding site for the sequential receptor oligomerization.     

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

Summary This study sheds new light on the role of acidic residues present in the active site cavity of human aromatase. Eight acidic residues (E129, D222, E245, E302, D309, E379, D380 and D476) lining the cavity are identified and studied using comparative modeling, docking, molecular dynamics as well as statistical techniques. The structural environment of these acidic residues is studied to assess the stability of the corresponding carboxylate anions. Results indicate that the environment of the residues E245, E302 and D222 is most suitable for carboxylate ion formation in the uncomplexed form. However, the stability of D309, D222 and D476 anions is seen to increase on complexation to steroidal substrates. In particular, the interaction between D309 and T310, which assists proton transfer, is found to be formed following androgen/nor-androgen complexation. The residue D309 is found to be clamped in the presence of substrate which is not observed in the case of the other residues although they exhibit changes in properties following substrate binding. Information entropic analysis indicates that the residues D309, D222 and D476 have more conformational flexibility compared to E302 and E245 prior to substrate binding. Interaction similar to that between D476 and D309, which is expected to assist androgen aromatization, is proposed between E302 and E245. The inhibition of aromatase activity by 4-hydroxy androstenedione (formestane) is attributed to a critical hydrogen bond formation between the hydroxy moiety and T310/D309 as well as the large distance from D476. The results corroborate well with earlier site directed mutagenesis studies.     

A novel reagent for the chemical phosphorylation of oligonucleotides

A novel phosphoramidite reagent was developed to convert terminal hydroxyl groups of oligonucleotides into phosphate monoesters. The reagent’s appearance as a solid foam is advantageous for its manipulation and handling in solid-phase synthesis and improves its thermal stability.     

PIFA-mediated synthesis of novel pyrazoloquinolin-4-ones as potential ligands for the estrogen receptor

A facile and efficient preparation of pyrazoloquinolin-4-ones, as potential ligands for the estrogen receptor, via a PIFA [phenyliodine(III)bis(trifluoroacetate)] promoted cyclization reaction with overall yields up to 29% over six steps is described. The employed strategy, based on an electrophilic amidation reaction as the key step of the synthesis, allows the generation of a diverse array of derivatives.     

Regioselective synthesis of 1,3,5-triaryl-4-alkylpyrazoles: novel ligands for the estrogen receptor

[reaction: see structure] A regioselective synthesis of 4-alkyl-1,3,5-triarylpyrazoles has been developed for the preparation of unsymmetrically substituted systems of interest as ligands for the estrogen receptor.     

Anticancer effects of brucine and gemcitabine combination in MCF-7 human breast cancer cells

This study was designed to investigate the combination effects of brucine and gemcitabine, each with anticancer properties, in MCF-7 human breast cancer cells in culture. With regard to cell viability, effects of both the drugs and their combinations were inversely proportional to dose and time. For various proportional drug combinations studied, combination effects were analysed using CompuSyn software. The analyses revealed synergistic and/or additive effects regarding cell viability, anchorage-independent growth and cell migration. Combination analyses exhibited diversified impacts of the type of combination treatment, namely pretreatment with either drug followed by exposure to the other, or treatment with both drugs at the same time. Compared with untreated cells, combination treatment of asynchronised MCF-7 cells resulted in 17.2 × decrease in G2 phase, increasing G1 (2.1 × ) and S (1.5 × ) phase cells in cell cycle analysis. Brucine, either individually or in combination, but not gemcitabine, inhibited NF-kB subunit (p65) expression in MCF-7 cells.     

Antiproliferative effect of 2-Hydroxy-6-tridecylbenzoic acid from ginkgo biloba sarcotestas through the aryl hydrocarbon receptor pathway in triple-negative breast cancer cells

Abstract

This study aims to isolate the potential antiproliferative and cytotoxic compounds from ginkgo biloba sarcotestas (GBS) and investigates the underlying mechanism in human MDA-MB-231 and mouse 4T-1 triple-negative breast cancer cells. Our results showed that 2-Hydroxy-6-tridecylbenzoic acid was isolated by cytotoxicity-guided fractionation where different fractions were assessed using MTT assay against MDA-MB-231 and 4T-1 cells. Colony formation assay showed that 2-Hydroxy-6-tridecylbenzoic acid significantly inhibited cell proliferation. The inhibition was associated with the enhancement of cytochrome P450 (CYP) 1B1 expression in a dose- and time-dependent manner and no significant change of CYP1A1 expression by qPCR and Western blot assays in MDA-MB-231 and 4T-1 cells. The mechanism was further demonstrated by the activation of aryl hydrocarbon receptor (AhR) pathway with the upregulation of AhR, AhR nuclear translocator (ARNT) and AhR-dependent xenobiotic response elements (XRE) activity. These findings may have implications for development of anticancer agents containing 2-Hydroxy-6-tridecylbenzoic acid as functional additives.     

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS)

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V_pp. Finally, under the applied voltage of 48 MHz–8 V_pp and a flow rate of 290 μm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.