Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve beta-catenin and cyclin D1 down-regulation

Exisulind and its analogues are inhibitors of cyclic GMP phosphodiesterases (PDEs) that have been shown to activate and induce protein kinase G, resulting in the induction of apoptosis in colon cancer cells. These drugs also reduce beta-catenin protein levels and decrease cyclin D1 mRNA levels in SW480 cells. Herein we report on studies pertaining to exisulind regulation of beta-catenin levels and activity in colon tumor cells. Exisulind and its higher-affinity PDE analogues, (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamide hydrochloride (CP461) and (Z)-1H-indene-3-acetamide, 5-fluoro-2-methyl-N-(phenylmethyl)-1-[(3,4,5-trimethoxyphenyl)methylene] (CP248), reduced beta-catenin, including the nuclear beta-catenin in SW480 cells (EC(50) approximately 200 microM, 1 microM, and <1 microM, respectively). The 50% reduction of beta-catenin was seen in 8-14 hr. There was no change in beta-catenin mRNA. Exisulind-induced beta-catenin reduction was blocked by the proteasomal inhibitor MG132 (Z-leu-Leu-Leu-CHO), indicating that the effect of exisulind involved ubiquitin-proteasomal degradation. A consequence of reduced beta-catenin in SW480 cells was that exisulind, CP461, and CP248 caused a concentration- and time-dependent decrease in cyclin D1 levels (EC(50) approximately 300 microM, 1 microM, and <1 microM, respectively) in 4 hr. The effect was via decreased cyclin D1 mRNA levels. Exisulind-induced degradation of beta-catenin was not blocked by the inhibition of caspase-3 activity and/or apoptosis, and some SW480 cells showed a reduction in beta-catenin levels before the appearance of early apoptosis indicators. Expression of the N-terminal 170 amino acid fragment of beta-catenin reduced the effects of beta-catenin degradation, cyclin D1 reduction, and the apoptosis response to exisulind. These results indicate that exisulind-induced beta-catenin degradation precedes the induction of apoptosis and that the down-regulation of inappropriate beta-catenin-activated genes accounts in part for the pro-apoptotic effects of exisulind and CP461 in colon tumor cells.     

Combined Effect of Low-Frequency Ultrasound and Iontophoresis: Applications for Transdermal Heparin Delivery

Pharmaceutical Research -     

The Effects of Permeation Enhancers on the Surface Morphology of the Rat Nasal Mucosa: A Scanning Electron Microscopy Study

A rat model has been developed to compare relative morphological changes in the nasal mucosa after exposure to potential membrane permeation enhancers. Scanning electron microscopy was used to characterize gross structural and specific cellular changes following exposure. Micrographs of the rat nasal mucosa were scored in four categories: (1) mucosal surface integrity, (2) ciliary morphology, (3) mucus/extracellular debris, and (4) presence of red blood cells. The order of increasing morphological damage resulting from a 5-min exposure to each surfactant was 0.5% Solulan C-24 0.5% Solulan C-24/0.5% sodium tauro-24,25-dihydrofusidate (STDHF) < 0.5% STDHF < 1.0% STDHF 1.0% Laureth-9 < 1.0% sodium taurodeoxycholate 1.0% sodium deoxycholate. The changes observed in the mucosal morphology after exposure to the various surfactants are in general agreement with data in the literature. This model is able to compare rapidly the relative morphological effects on the mucosal membrane of different nasal formulations.     

Involvement of metabotropic glutamate receptors in Gi- and Gs-dependent modulation of adenylate cyclase activity induced by a novel cognition enhancer NS-105 in rat brain

The effect of a novel cognition enhancer [(+)-5-oxo-

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-prolinepiperidinamide monohydrate] (NS-105) on cAMP formation was investigated in both slices and membranes of the rat cerebral cortex. NS-105 (10⁻⁸–10⁻⁶ M) inhibited forskolin-stimulated cAMP formation in membranes, however, the compound significantly enhanced the cAMP formation in pertussis toxin-pre-treated membranes, an action that was abolished by cholera toxin. In contrast, in digitonin-permeabilized membranes, NS-105 had no influence on Mn²⁺-stimulated cAMP formation. Both of the inhibitory and facilitatory actions of NS-105 on cAMP formation were mimicked by a metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and an adrenergic α₂ agonist UK-14,304, and blocked by a mGluR antagonist 2-amino-3-phosphonopropanoate but not by an α₂ antagonist yohimbine. In cortical slices, NS-105 (10⁻⁸–10⁻⁷ M) inhibited forskolin-stimulated cAMP accumulation but enhanced isoproterenol-stimulated cAMP accumulation, as did by a GABA_B agonist (−)baclofen. On the other hand, (−)baclofen, while it significantly inhibited cAMP accumulation in slices, did no longer inhibit cAMP accumulation, when treated with NS-105 (10⁻⁸–10⁻⁵ M). Similarly, (−)baclofen-induced inhibition of the cAMP accumulation was reversed by 1S,3R-ACPD and UK-14,304. NS-105 (10⁻⁶) increased [³⁵S]GTPγS binding in the intact but not digitonin-permeabilized cortical membranes, as produced by UK-14,304, although the compound (10⁻⁹–10⁻³ M) had no influence on various neurotransmitter receptor bindings, including α₂ receptors. These results suggest that NS-105 modulates adenylate cyclase activity by stimulating mGluRs which might coupled to both G_i/G_o and G_s. © 1997 Elsevier Science B.V. All rights reserved.     

髓母细胞瘤组织MEF2C表达与预后相关性分析

目的：探讨肌细胞增强因子2C（myoeyte enhancer factor2C,MEF2C）表达情况与髓母细胞瘤患者预后的关系。方法：选取3家三级甲等医院2001-01—01—2007—12-31病理确诊的髓母细胞瘤患者56例,其中泰安市中心医院12例．齐鲁医院26例,山东省立医院18例;对照组脑组织取自泰安市中心医院2010-01-01-2010—12—3112例非肿瘤脑外伤患者。免疫组织化学法检测56例髓母细胞瘤患者肿瘤组织和12例非肿瘤患者脑组织中MEF2C的表达;收集髓母细胞瘤患者临床资料,应用统计学方法对髓母细胞瘤患者进行生存分析。结果：髓母细胞瘤患者肿瘤组织MEF2C相对高表达率为53．6％（30／56）,非肿瘤患者脑组织MEF2C相对高表达率为0（0／12）,两组比较差异有统计学意义,χ2=11．50,P=0．001;肿瘤患者肿瘤组织MEF2C相对高表达组与相对低表达组间生存时间的差异有统计学意义,χ2=4．34,P=0．037。MEF2C相对高表达（p=0．023）、肿瘤转移（P=0．042）与患者3年生存率降低显著有关,而患者性别、年龄、肿瘤大小、肿瘤部位与患者3年生存率无关,P值均〉0．05。结论：MEF2C在患者肿瘤组织中的高表达可降低患者3年生存率,是患者预后不良的危险因素之一。     

N-Acetylcysteine Rinse for Thick Secretion and Mucositis of Head and Neck Chemoradiotherapy (Alliance MC13C2): A Double-Blind Randomized Clinical Trial

ObjectiveTo determine whether N-acetylcysteine rinse was safe and could improve thickened secretions and dry mouth during and after radiotherapy.Patients and MethodsWe designed a prospective pilot double-blind, placebo-controlled randomized clinical trial (Alliance MC13C2). Adult patients (age ≥18 years) were enrolled if they underwent chemoradiotherapy (≥60 Gy). Patients initiated testing rinse within 3 days of starting radiotherapy. With swish-and-spit, they received 10% N-acetylcysteine (2500 mg daily) or placebo rinse solution 5 times daily during radiotherapy and 2 weeks postradiotherapy. The primary aim was to evaluate N-acetylcysteine in improvement of saliva viscosity with the Groningen Radiotherapy-Induced Xerostomia questionnaire. Secondary aims included evaluating xerostomia improvement by the same questionnaire and with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire–Head and Neck-35 Questions survey and adverse-event profiles. The type I error rate was 20%.ResultsThirty-two patients undergoing chemoradiotherapy were enrolled. Baseline characteristics were balanced for placebo (n=17) and N-acetylcysteine (n=15). N-acetylcysteine was better for improving sticky saliva (area under curve, P=.12). Scores of multiple secondary end points favored N-acetylcysteine, including sticky saliva daytime (P=.04), daytime and total xerostomia (both P=.02), pain (P=.18), and trouble with social eating (P=.15). Repeated measures models confirmed the findings. Taste was a major dissatisifer for N-acetylcysteine rinse; however, both testing rinses were safe and well tolerated overall.ConclusionOur pilot data showed that N-acetylcysteine rinse was safe and provided strong evidence of potential efficacy for improving thickened saliva and xerostomia by patient-reported outcome. A confirmatory phase 3 trial is required.Trial Registrationclinicaltrials.gov Identifier: NCT02123511