Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.This research was funded by the Prostate Cancer Research Centre charity (registered UK charity no. 1156027), Grant Number AA1. A small financial contribution was also made through intra-mural funds from the Royal Veterinary College.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.016110

Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at αxβ3γ2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of α1 subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position -6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (LDi region) of GABAA/Bz receptors (see 32 and 33). Moreover, substituents located at position -3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L1, while N(2) presumably underwent a hydrogen bonding interaction with H1. Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were “near GABA neutral antagonists”. Based on the SAR, the most potent (in vitro) α1 selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.1–3 These data prompted the synthesis of the β-carbolines presented here

Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium Induced Calcium Release Mechanism

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i. We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Kinetics modeling and occupancy studies of a novel C-11 PET tracer for VAChT in nonhuman primates

INTRODUCTION: Deficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (−)-[(11)C]TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain are reported. METHODS: MicroPET brain imaging of (−)-[(11)C]TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (−)-vesamicol, or S-(−)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (−)-vesamicol. RESULTS: Baseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (V(T)) values derived from both 2TCM and Logan analysis with blood input revealed ~3-fold higher levels of (−)-[(11)C]TZ659 in the striatum than the cerebellar hemispheres. Injection of (−)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (−)-[(11)C]TZ659. In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S-(−)-eticlopride, a dopamine D(2)–like receptor antagonist, increased striatal uptake of (−)-[(11)C]TZ659. Striatal binding potential (BP(ND), range of 0.33 – 1.6 with cerebellar hemispheres as the reference region) showed good correlation (r(2) = 0.97) between SRTM and LoganREF. Occupancy studies found that ~ 0.0057 mg/kg (−)-vesamicol produced 50% VAChT occupancy in the striatum. CONCLUSION: (−)-[(11)C]TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain

Multi-labeled immunofluorescence for CD1, c-Myc and FRA1.

<p>Co-expression of three proteins CD1 (FITC-green), c-Myc (Cy3-red) and FRA1 (Cy5-cyan) in normal (A, B and C) and malignant feline oral tissue cores (D, E and F) and DAPI, counterstain is blue; images are representative tissue cores from the tissue array with over 200 samples. Whole tissue cores (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#pone.0161103.s004" target="_blank">S4 Fig</a>) were imaged using a Zeiss Axioscan Z.1 slide scanner (Carl Zeiss) at 20x magnification with a Calibri.2 LED lights and integration times of the Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics) and fluorescent signals were optimized at the start of study so as to not oversaturate the signal for each antibody. For quantitative co-localization a randomly selected area was imaged using an Olympus IX81 confocal system and a dry 40x objective (A, normal and D, malignant); these areas were further magnified (6x digital zoom) and re-imaged (B, normal and E, malignant). The resulting images were deconvolved using Huygens Software (C, normal and F, malignant) for the calculation of GIC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#sec002" target="_blank">Methods</a>). Scale bar = 10μm.</p

Box plots for differential co-localization of CD1, c-MYC and FRA1 in feline tissue samples.

<p>High magnification fluorescent images (e.g. as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#pone.0161103.g004" target="_blank">Fig 4</a>) of feline tissue cores (normal, gray outline; squamous cell carcinoma, brown outlined boxes) were deconvolved and used for the calculation of Global Intersection Coefficient (GIC) using Huygens software. 19 individual tissue samples were used for the three proteins and nuclear stain DAPI (DP) to measure the co-localization of DP/CD1 (blue/green bars), DP/c-MYC (blue/red), DP/FRA1 (blue/cyan), CD1/c-MYC (green/red), CD1/FRA1 (green/cyan) and c-MYC/FRA1 (red/cyan) using GIC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#sec002" target="_blank">Materials and Methods</a>). Significance of difference in the GIC for co-localization between normal and FOSCC was calculated using the Mann Whitney U test (* = <i>p</i><0.01, ns = not significant).</p