225 patients from the TAM arm of 89-30-52.

<p>225 patients from the TAM arm of 89-30-52.</p

GREB1 is localized in the nucleus in ER-positive breast cancer cell line and breast cancer tissues.

<p>Subcellular localization of GREB1 in breast cancer cells was determined by immunofluorescent microscopy and immunohistochemical staining (IHC). A to C, E2-deprived MCF-7 cells were stimulated with E2 for 24 h and then stained for GREB1 in red (Alexa Fluor 555, A) and nucleus in blue (DAPI, B), and C gave the merged picture. GREB1 is almost exclusively localized in the nucleus. D to F showed staining of negative control lacking primary GREB1 antibody. The nuclear localization of GREB1 was further confirmed by introducing exogenous GREB1 into MCF-7 cells. GREB1 was stained in red in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3G</a>, nucleus was stained in blue in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3H</a>, the merged image was shown as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3I</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3J to 3L</a> showed the staining of negative control. IHC staining showed that ER-positive MCF-7 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3M</a>) and ER-positive breast cancer tissues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3Q</a>) detected GREB1 expression (brown). ER-negative breast cancer cell line MDA-MB-231 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3O</a>) and ER-negative breast cancer tissues expressed undetectable levels of GREB1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3R</a>). HE staining was also showed for ER-positive breast cancer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3N</a>) and ER-negative breast cancer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g003" target="_blank">Figure 3P</a>).</p

IL6/STAT3 modulates estrogen-induced GREB1 transcriptional activity in breast cancer cells.

<p>E2 treatment increases GREB1 mRNA levels by approximately 43-fold after 48 h treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5A</a>). ICI abolishes E2-induced GREB1 expression. IL-6 has an inhibitory effect on GREB1 expression, which inhibits GREB1 expression by approximately 44% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5A</a>). Transfection with STAT3 siRNA (SiSTAT3) duplexes increased E2 induced expression of GREB1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5B</a>) whereas transfection with constitutively active STAT3 vector (STAT3-C) suppressed E2 induced expression of GREB1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5C</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5D to 5G</a>, MCF-7 cells were estrogen deprived for 3 days before transfection with 0.5 µg luciferase GREB1 promoter-reporter construct and 0.1 ug phRL-SV40 Renilla internal control. The following day, transfected cells were refed with medium containing 1 nM E2 and 10 ng/ml IL-6 for a further 24 h before lysis and measurement of luciferase activity. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5D, E</a>2 treatment increases luciferase activity by approximately 37-fold over the control. Estrogen receptor antagonist ICI blocks the E2-mediated GREB1 induction. IL-6 inhibits E2-induced GREB1 transcriptional activity by approximately 38%. To determine the best efficient concentration for IL-6, hormone-starved MCF-7 cells were treated with various concentrations of IL-6 (0∼100 ng/ml) for 24 h. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5E</a> shows that the maximum reduction of E2-mediated GREB1 activity is seen at 10 ng/ml, the inhibitory effects are decreased regardless of whether the doses are further increased or decreased throughout the experiment. In time course experiments, cells were treated with 10 ng/ml IL-6 at indicated time,. Pretreatment with IL-6 for less than half an hour dampered the antagonist function of IL-6 on suppression of E2-induced GREB1 greatly (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5F</a>). MCF-7 cells were then cotransfected with a GREB1 luciferase reporter construct, a STAT3 constitutively expressing vector (STAT3-C) and control vector respectively. The samples were treated with E2 as indicated before assaying for luciferase activity. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g005" target="_blank">Figure 5G</a>, STAT3-C decreases GREB1 transcription by approximately 39%. Data are shown as mean ± SD. *<i>P</i><0.05; **<i>P</i><0.01.</p

GREB1 is induced by E2 in ER-positive breast cancer cell lines.

<p>A, Western blotting detects a single band of ∼216 kD in E2-deprived MCF-7 cells treated with estrogen for 24 and 48 hrs while no protein is detected in the ER-negative BT-549 cells or in MCF-7 cells grown in estrogen-free conditions. B, GREB1 protein expression was reduced in the MCF-7 cells treated with estrogen plus ICI 182,780 (ICI) compared to that observed in cells treated with estrogen alone. C, Figure C shows loss of detectable GREB1 protein when GREB1 is knocked down by GREB1 siRNA (SiGREB1) at 48 hours. Control siRNA (CSiRNA) has no effect on E2-induced GREB1 production. Corresponding densitometric analysis of the bands performed with the ImageQuant program (Bio-Rad) were shown below the Western blot. E2-induced GREB1mRNA levels were also analyzed, GREB1 mRNA levels were well correlated with GREB1 protein expressions. D, GREB1 mRNA is notable as early as 24 hours and lasts up to 48 hours as presented in a time course study. BT-549 cells express GREB1 mRNA as low as the control. E and F, ICI treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g001" target="_blank">Figure 1E</a>) and silencing the GREB1 gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g001" target="_blank">Figure 1F</a>) significantly reduced the GREB1 expression at transcriptional level. Data are shown as mean ± SD. *<i>P</i><0.05; **<i>P</i><0.01.</p

STAT3 physically interacts with ERα.

<p>To confirm whether there is a physical interaction between STAT3 and ERα, we transfected 293T cells with FLAG-tagged STAT3-C (2 µg) alone or together with ERα (2 µg). 48 h after transfection, cells were treated with 1 nM E2 for 3 h. Cell lysates were immunoprecipited with anti-ERα and immunoblotted with anti-FLAG as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g006" target="_blank">Figure 6A</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g006" target="_blank">Figure 6B</a> showed that cell lysates were first immunoprecipitated with anti-FLAG from two different commercially available resources (Millipore and Sigma) respectively followed by immunoblotted with anti-ERα. The results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046410#pone-0046410-g006" target="_blank">Figure 6</a> indicate that STAT3 indeed directly interacts with ERα.</p

GREB1 mRNA correlates with ER status in breast cancer patients, and predicts responses in Tamoxifen-treated patients.

<p>Meta-analysis was performed using the Oncomine Research Platform- based microarray studies. A. Microarray analysis of 7 normal breast tissues (red bar) and 40 breast cancers (blue bar). The mean values of GREB1 mRNA expression are shown here. B. Microarray analysis of 2321 patients (1651 ER-positive, 670 ER-negative). The level of GREB1 expression is higher in ER-positive breast cancer patients, which are depicted by red-colored bars and ER-negative patients are represented by blue ones. Kaplan-Meier survival plots demonstrate the prognostic relevance of GREB1 expression on patient survival using various data cohorts. C. Reduced expression of GREB1 is predictive of lower relapse free survival (HR = 0.66, <i>p</i> = 1.1×10⁻¹⁰) for all patients; D. For ER+ breast cancer patients, lower GREB1 expression is correlated with decreased relapse free survival (HR = 0.75, <i>p</i> = 0.0029); E. Endocrine treated ER+ breast cancer patients have reduced relapse free survival if their tumors expressed lower GREB1 level (HR = 0.63, <i>p</i> = 0.041).</p

Point-of-Care Determination of Acetaminophen Levels with Multi-Hydrogen Bond Manipulated Single-Molecule Recognition (eMuHSiR)

This work aims to face the challenge of monitoring small molecule drugs accurately and rapidly for point-of-care (POC) diagnosis in current clinical settings. Overdose of acetaminophen (AP), a commonly used over the counter (OTC) analgesic drug, has been determined to be a major cause of acute liver failure in the US and the UK. However, there is no rapid and accurate detection method available for this drug in the emergency room. The present study examined an AP sensing strategy that relies on a previously unexplored strong interaction between AP and the arginine (Arg) molecule. It was found that as many as 4 hydrogen bonds can be formed between one Arg molecule and one AP molecule. By taking advantages of this structural selectivity and high tenability of hydrogen bonds, Arg, immobilized on a graphene surface via electrostatic interactions, was utilized to structurally capture AP. Interestingly, bonded AP still remained the perfect electrochemical activities. The extent of Arg–AP bonds was quantified using a newly designed electrochemical (EC) sensor. To verify the feasibility of this novel assay, based on multihydrogen bond manipulated single-molecule recognition (eMuHSiR), both pharmaceutical and serum sample were examined. In commercial tablet measurement, no significant difference was seen between the results of eMuHSiR and other standard methods. For measuring AP concentration in the mice blood, the substances in serum, such as sugars and fats, would not bring any interference to the eMuHSiR in a wide concentration range. This eMuHSiR method opens the way for future development of small molecule detection for the POC testing

Structure–Activity Relationship and Pharmacokinetic Studies of 1,5-Diheteroarylpenta-1,4-dien-3-ones: A Class of Promising Curcumin-Based Anticancer Agents

Forty-three 1,5-diheteroaryl-1,4-pentadien-3-ones were designed as potential curcumin mimics, structurally featuring a central five-carbon dienone linker and two identical nitrogen-containing aromatic rings. They were synthesized using a Horner–Wadsworth–Emmons reaction as the critical step and evaluated for their cytotoxicity and antiproliferative activities toward both androgen-insensitive and androgen-sensitive prostate cancer cell lines and an aggressive cervical cancer cell line. Most of the synthesized compounds showed distinctly better in vitro potency than curcumin in the four cancer cell lines. The structure–activity data acquired from the study validated (1<i>E</i>,4<i>E</i>)-1,5-dihereroaryl-1,4-pentadien-3-ones as an excellent scaffold for in-depth development for clinical treatment of prostate and cervical cancers. 1-Alkyl-1<i>H</i>-imidazol-2-yl, ortho pyridyl, 1-alkyl-1<i>H</i>-benzo­[<i>d</i>]­imidazole-2-yl, 4-bromo-1-methyl-1<i>H</i>-pyrazol-3-yl, thiazol-2-yl, and 2-methyl-4-(trifluoromethyl)­thiazol-5-yl were identified as optimal heteroaromatic rings for the promising in vitro potency. (1<i>E</i>,4<i>E</i>)-1,5-Bis­(2-methyl-4-(trifluoromethyl)­thiazol-5-yl)­penta-1,4-dien-3-one, featuring thiazole rings and trifluoromethyl groups, was established as the optimal lead compound because of its good in vitro potency and attractive in vivo pharmacokinetic profiles

Discovery of a Series of Thiazole Derivatives as Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion

Effective inhibitors of cancer cell migration and invasion can potentially lead to clinical applications as a therapy to block tumor metastasis, the primary cause of death in cancer patients. To this end, we have designed and synthesized a series of thiazole derivatives that showed potent efficacy against cell migration and invasion in metastatic cancer cells. The most effective compound, <b>5k</b>, was found to have an IC₅₀ value of 176 nM in the dose-dependent transwell migration assays in MDA-MB-231cells. At a dose of 10 μM, <b>5k</b> also blocked about 80% of migration in HeLa and A549 cells and 60% of invasion of MDA-MB-231 cells. Importantly, the majority of the derivatives exhibited no apparent cytotoxicity in the clonogenic assays. The low to negligible inhibition of cell proliferation is a desirable property of these antimigration derivatives because they hold promise of low toxicity to healthy cells as potential therapeutic agents. Mechanistic studies analyzing the actin cytoskeleton by microscopy demonstrate that compound <b>5k</b> substantially reduced cellular f-actin and prevented localization of fascin to actin-rich membrane protrusions. These results suggest that the antimigration activity may result from impaired actin structures in protrusions that are necessary to drive migration

Differentially expressed proteins in MSKE-treated and control cultures.

<p>Differentially expressed proteins in MSKE-treated and control cultures.</p