Stabilization of nontoxic Ajβ-oligomers: Insights into the mechanism of action of hydroxyquinolines in alzheimer’s disease

©2015 the authors. The extracellular accumulation of amyloid β (A/β) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aβ, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from A/β:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aβ fibrillar polymerization and direct depolymerization of existing Aβ fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aβ and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aβ associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aβ with an affinity of 1-10 μam and suppress the formation of large (>30kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aβ toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aβ oligomer formation through stabilization of small (dimeric) nontoxic Aβ conformers

Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney

The antiapoptotic Livin/ML-IAP gene has recently gained much attention as a potential new target for cancer therapy. Reports indicating that livin is expressed almost exclusively in tumours, but not in the corresponding normal tissue, suggested that the targeted inhibition of livin may present a novel tumour-specific therapeutic strategy. Here, we compared the expression of livin in renal cell carcinoma and in non-tumorous adult kidney tissue by quantitative real-time reverse transcription-PCR, immunoblotting, and immunohistochemistry. We found that livin expression was significantly increased in tumours (P=0.0077), but was also clearly detectable in non-tumorous adult kidney. Transcripts encoding Livin isoforms α and β were found in both renal cell carcinoma and normal tissue, without obvious qualitative differences. Livin protein in renal cell carcinoma samples exhibited cytoplasmic and/or nuclear staining. In non-tumorous kidney tissue, Livin protein expression was only detectable in specific cell types and restricted to the cytoplasm. Thus, whereas the relative overexpression of livin in renal cell carcinoma indicates that it may still represent a therapeutic target to increase the apoptotic sensitivity of kidney cancer cells, this strategy is likely to be not tumour-specific

Attenuation of doxorubicin-induced cardiotoxicity by mdivi-1: a mitochondrial division/mitophagy inhibitor

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties

A single-blinded trial of methotrexate versus azathioprine as steroid-sparing agents in generalized myasthenia gravis

<p>Abstract</p> <p>Background</p> <p>Long-term immunosuppression is often required in myasthenia gravis (MG). There are no published trials using methotrexate (MTX) in MG. The steroid-sparing efficacy of azathioprine (AZA) has been demonstrated after 18-months of starting therapy. However, AZA is considered expensive in Africa. We evaluated the steroid-sparing efficacy of MTX (17.5 mg weekly) compared with AZA (2.5 mg/kg daily) in subjects recently diagnosed with generalized MG by assessing their average monthly prednisone requirements.</p> <p>Methods</p> <p>The primary outcome was the average daily prednisone requirement by month between the two groups. Prednisone was given at the lowest dose to manage MG symptoms and adjusted as required according to protocol. Single-blinded assessments were performed 3-monthly for 2-years to determine the quantitative MG score and the MG activities of daily living score in order to determine those with minimal manifestations of MG.</p> <p>Results</p> <p>Thirty-one subjects (AZA n = 15; MTX n = 16) satisfied the inclusion criteria but only 24 were randomized. Baseline characteristics were similar. There was no difference between the AZA- and MTX-groups in respect of prednisone dosing (apart from months 10 and 12), in quantitative MG Score improvement, proportions in sustained remission, frequencies of MG relapses, or adverse reactions and/or withdrawals. The MTX-group received lower prednisone doses between month 10 (p = 0.047) and month 12 (p = 0.039). At month 12 the prednisone dose per kilogram bodyweight in the MTX-group (0.15 mg/kg) was half that of the AZA-group (0.31 mg/kg)(p = 0.019).</p> <p>Conclusions</p> <p>This study provides evidence that in patients with generalized MG methotrexate is an effective steroid-sparing agent 10 months after treatment initiation. Our data suggests that in generalized MG methotrexate has similar efficacy and tolerability to azathioprine and may be the drug of choice in financially constrained health systems.</p> <p>Trial registration</p> <p>SANCTR:DOH-27-0411-2436</p

Trends in incidence of childhood cancer in Australia, 1983–2006

Cancer risk is increased substantially in adult kidney transplant recipients, but the long-term risk of cancer in childhood recipients is unclear. Using the Australian and New Zealand Dialysis and Transplant Registry, the authors compared overall and site-specific incidences of cancer after transplantation in childhood recipients with population-based data by using standardized incidence ratios (SIRs). Among 1734 childhood recipients (median age 14 years, 57% male, 85% white), 289 (16.7%) developed cancer (196 nonmelanoma skin cancers, 143 nonskin cancers) over a median follow-up of 13.4 years. The 25-year cumulative incidences of any cancer were 27% (95% confidence intervals 24-30%), 20% (17-23%) for nonmelanoma skin cancer, and 14% (12-17%) for nonskin cancer (including melanoma). The SIR for nonskin cancer was 8.23 (95% CI 6.92-9.73), with the highest risk for posttransplant lymphoproliferative disease (SIR 45.80, 95% CI 32.71-62.44) and cervical cancer (29.4, 95% CI 17.5-46.5). Increasing age at transplantation (adjusted hazard ratio [aHR] per year 1.10, 95% CI 1.06-1.14), white race (aHR 3.36, 95% CI 1.61-6.79), and having a functioning transplant (aHR 2.27, 95% CI 1.47-3.71) were risk factors for cancer. Cancer risk, particularly for virus-related cancers, is increased substantially after kidney transplantation during childhood

Zinc-Chelation Contributes to the Anti-Angiogenic Effect of Ellagic Acid on Inhibiting MMP-2 Activity, Cell Migration and Tube Formation

Ellagic acid (EA), a dietary polyphenolic compound, has been demonstrated to exert anti-angiogenic effect but the detailed mechanism is not yet fully understood. The aim of this study was to investigate whether the zinc chelating activity of EA contributed to its anti-angiogenic effect.The matrix metalloproteinases-2 (MMP-2) activity, a zinc-required reaction, was directly inhibited by EA as examined by gelatin zymography, which was reversed dose-dependently by adding zinc chloride. In addition, EA was demonstrated to inhibit the secretion of MMP-2 from human umbilical vein endothelial cells (HUVECs) as analyzed by Western blot method, which was also reversed by the addition of zinc chloride. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), known to down-regulate the MMP-2 activity, was induced by EA at both the mRNA and protein levels which was correlated well with the inhibition of MMP-2 activity. Interestingly, zinc chloride could also abolish the increase of EA-induced RECK expression. The anti-angiogenic effect of EA was further confirmed to inhibit matrix-induced tube formation of endothelial cells. The migration of endothelial cells as analyzed by transwell filter assay was suppressed markedly by EA dose-dependently as well. Zinc chloride could reverse these two effects of EA also in a dose-dependent manner. Since magnesium chloride or calcium chloride could not reverse the inhibitory effect of EA, zinc was found to be involved in tube formation and migration of vascular endothelial cells.Together these results demonstrated that the zinc chelation of EA is involved in its anti-angiogenic effects by inhibiting MMP-2 activity, tube formation and cell migration of vascular endothelial cells. The role of zinc was confirmed to be important in the process of angiogenesis

Nematode and Arthropod Genomes Provide New Insights into the Evolution of Class 2 B1 GPCRs

Nematodes and arthropods are the most speciose animal groups and possess Class 2 B1 G-protein coupled receptors (GPCRs). Existing models of invertebrate Class 2 B1 GPCR evolution are mainly centered on Caenorhabditis elegans and Drosophila melanogaster and a few other nematode and arthropod representatives. The present study reevaluates the evolution of metazoan Class 2 B1 GPCRs and orthologues by exploring the receptors in several nematode and arthropod genomes and comparing them to the human receptors. Three novel receptor phylogenetic clusters were identified and designated cluster A, cluster B and PDF-R-related cluster. Clusters A and B were identified in several nematode and arthropod genomes but were absent from D. melanogaster and Culicidae genomes, whereas the majority of the members of the PDF-R-related cluster were from nematodes. Cluster A receptors were nematode and arthropod-specific but shared a conserved gene environment with human receptor loci. Cluster B members were orthologous to human GCGR, PTHR and Secretin members with which they probably shared a common origin. PDF-R and PDF-R related clusters were present in representatives of both nematodes and arthropods. The results of comparative analysis of GPCR evolution and diversity in protostomes confirm previous notions that C. elegans and D. melanogaster genomes are not good representatives of nematode and arthropod phyla. We hypothesize that at least four ancestral Class 2 B1 genes emerged early in the metazoan radiation, which after the protostome-deuterostome split underwent distinct selective pressures that resulted in duplication and deletion events that originated the current Class 2 B1 GPCRs in nematode and arthropod genomes.This work was supported by the Portuguese Foundation for Science and Technology (FCT) project PTDC/BIA-BCM/114395/2009, by the European Regional Development Fund through COMPETE and FCT under the project ‘‘PEst-C/MAR/LA0015/2011.’’ RCF is in receipt of an FCT grant (SFRH/BPD/89811/2012) and JCRC is supported by auxiliary research contract FCT Pluriannual funds attributed to CCMAR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

<p>Abstract</p> <p>Background</p> <p>Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells.</p> <p>Methods</p> <p>The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth <it>in vivo </it>were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression.</p> <p>Results</p> <p>BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10.</p> <p>Conclusions</p> <p>These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.</p

MicroRNA Expression Variability in Human Cervical Tissues

MicroRNAs (miRNAs) are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Deregulation of miRNA expression has been discovered in a wide variety of tumours and it is now clear that they contribute to cancer development and progression. Cervical cancer is one of the most common cancers in women worldwide and there is a strong need for a non-invasive, fast and efficient method to diagnose the disease. We investigated miRNA expression profiles in cervical cancer using a microarray platform containing probes for mature miRNAs. We have evaluated miRNA expression profiles of a heterogeneous set of cervical tissues from 25 different patients. This set included 19 normal cervical tissues, 4 squamous cell carcinoma, 5 high-grade squamous intraepithelial lesion (HSIL) and 9 low-grade squamous intraepithelial lesion (LSIL) samples. We observed high variability in miRNA expression especially among normal cervical samples, which prevented us from obtaining a unique miRNA expression signature for this tumour type. However, deregulated miRNAs were identified in malignant and pre-malignant cervical tissues after tackling the high expression variability observed. We were also able to identify putative target genes of relevant candidate miRNAs. Our results show that miRNA expression shows natural variability among human samples, which complicates miRNA data profiling analysis. However, such expression noise can be filtered and does not prevent the identification of deregulated miRNAs that play a role in the malignant transformation of cervical squamous cells. Deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of this tumour type

The Neuropeptide PDF Acts Directly on Evening Pacemaker Neurons to Regulate Multiple Features of Circadian Behavior

Animals use distinct sets of clock neurons to time behaviors in the morning and evening. In this article, the direct neural targets for morning neurons and the neuropeptide pigment dispersing factor are revealed in the fruit fly