Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD(+) catabolizing enzyme

Schistosomiasis is a major tropical parasitic disease. For its treatment, praziquantel remains the only effective drug available and the dependence on this sole chemotherapy emphasizes the urgent need for new drugs to control this neglected disease. In this context, the newly characterized Schistosoma mansoni NAD(+) catabolizing enzyme (SmNACE) represents a potentially attractive drug target. This potent NAD(+)glycohydrolase, which is localized to the outer surface (tegument) of the adult parasite, is presumably involved in the parasite survival by manipulating the host's immune regulatory pathways. In an effort to identify SmNACE inhibitors, we have developed a sensitive and robust fluorometric high-throughput screening assay. The implementation of this assay to the screening of a highly diverse academic chemical library of 14,300 molecules yielded, after secondary assays and generation of dose-response curves, the identification of two natural product inhibitors, cyanidin and delphinidin. These confirmed hits inhibit SmNACE with IC(50) values in the low micromolar range. To rationalize the structure-activity relationship, several related flavonoids were tested, thereby leading to the identification of 15 additional natural product inhibitors. A selection of representative flavonoid inhibitors indicated that although they also inhibit the homologous human CD38, a selectivity in favor of SmNACE could be reached. Docking studies indicated that these inhibitors mimic the binding mode of the enzyme substrate NAD(+) and suggested the pharmacophoric features required for SmNACE active site recognition.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

Targeted liposomes: convenient coupling of ligands to preformed vesicles using "click chemistry"

An efficient and convenient chemoselective conjugation method based on "click chemistry" was developed for coupling ligands to the surface of preformed liposomes. It can be performed under mild conditions in aqueous buffers; the use of a water soluble Cu(I) chelator, such as bathophenanthrolinedisulfonate, was essential to obtain good yields in reasonable reaction times. A model reaction was achieved in which, in a single step, an unprotected alpha-D-mannosyl derivative carrying a spacer arm functionalized with an azide group was conjugated to the surface of vesicles presenting a synthetic lipid carrying a terminal alkyne function. When liposomes composed of saturated phospholipids were used, the reaction conditions developed in the present work did not damage the membranes as measured by the absence of leakage of entrapped 5,6-carboxyfluorescein. Moreover, as assessed by agglutination experiments using concanavalin A, the mannose residues were perfectly accessible on the surface of the targeted vesicles.     

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

Background

The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)₂D₃, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)₂D₃ downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/Findings

To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)₂D₃ treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/Significance

These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.     

Receptor for advanced glycation end-products (RAGE) modulates neutrophil adhesion and migration on glycoxidated extracellular matrix

AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.     

Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling and Up-regulation of Egr-1 in Hypoxic Macrophages

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C βII, ERK1/2, and c-Jun NH₂-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.     

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.