Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.</p> <p>Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.</p> <p>Methods</p> <p>We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.</p> <p>Results</p> <p>Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness.</p> <p>Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.</p> <p>Conclusion</p> <p>a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;</p> <p>b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;</p> <p>c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.</p

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets

In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

The treatment of Trypanosoma cruzi infection (the cause of human Chagas disease) remains a significant challenge. Only two drugs, both with substantial toxicity, are available and the efficacy of these dugs is often questioned – in many cases due to the limitations of the methods for assessing efficacy rather than to true lack of efficacy. For these reasons relatively few individuals infected with T. cruzi actually have their infections treated. In this study, we report on innovative methods that will facilitate the discovery of new compounds for the treatment of T. cruzi infection and Chagas disease. Utilizing fluorescent and bioluminescent parasite lines, we have developed in vitro tests that are reproducible and facile and can be scaled for high-throughput screening of large compound libraries. We also validate an in vivo screening test that monitors parasite replication at the site of infection and determines the effectiveness of drug treatment in less than two weeks. More importantly, results in this rapid in vivo test show strong correlations with those obtained in long-term (e.g. 40 day or more) treatment assays. The results of this study remove one of the obstacles for identification of effective and safe compounds to treat Chagas disease

Phenotype Recognition with Combined Features and Random Subspace Classifier Ensemble

<p>Abstract</p> <p>Background</p> <p>Automated, image based high-content screening is a fundamental tool for discovery in biological science. Modern robotic fluorescence microscopes are able to capture thousands of images from massively parallel experiments such as RNA interference (RNAi) or small-molecule screens. As such, efficient computational methods are required for automatic cellular phenotype identification capable of dealing with large image data sets. In this paper we investigated an efficient method for the extraction of quantitative features from images by combining second order statistics, or Haralick features, with curvelet transform. A random subspace based classifier ensemble with multiple layer perceptron (MLP) as the base classifier was then exploited for classification. Haralick features estimate image properties related to second-order statistics based on the grey level co-occurrence matrix (GLCM), which has been extensively used for various image processing applications. The curvelet transform has a more sparse representation of the image than wavelet, thus offering a description with higher time frequency resolution and high degree of directionality and anisotropy, which is particularly appropriate for many images rich with edges and curves. A combined feature description from Haralick feature and curvelet transform can further increase the accuracy of classification by taking their complementary information. We then investigate the applicability of the random subspace (RS) ensemble method for phenotype classification based on microscopy images. A base classifier is trained with a RS sampled subset of the original feature set and the ensemble assigns a class label by majority voting.</p> <p>Results</p> <p>Experimental results on the phenotype recognition from three benchmarking image sets including HeLa, CHO and RNAi show the effectiveness of the proposed approach. The combined feature is better than any individual one in the classification accuracy. The ensemble model produces better classification performance compared to the component neural networks trained. For the three images sets HeLa, CHO and RNAi, the Random Subspace Ensembles offers the classification rates 91.20%, 98.86% and 91.03% respectively, which compares sharply with the published result 84%, 93% and 82% from a multi-purpose image classifier WND-CHARM which applied wavelet transforms and other feature extraction methods. We investigated the problem of estimation of ensemble parameters and found that satisfactory performance improvement could be brought by a relative medium dimensionality of feature subsets and small ensemble size.</p> <p>Conclusions</p> <p>The characteristics of curvelet transform of being multiscale and multidirectional suit the description of microscopy images very well. It is empirically demonstrated that the curvelet-based feature is clearly preferred to wavelet-based feature for bioimage descriptions. The random subspace ensemble of MLPs is much better than a number of commonly applied multi-class classifiers in the investigated application of phenotype recognition.</p

A Cell-Based Small Molecule Screening Method for Identifying Inhibitors of Epithelial-Mesenchymal Transition in Carcinoma

Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma

Resolving the Role of Actoymyosin Contractility in Cell Microrheology

Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells—stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM) and particle-tracking microrheology measurements of living cells

A Unique Role for Nonmuscle Myosin Heavy Chain IIA in Regulation of Epithelial Apical Junctions

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis

Anticancer potential of Thevetia peruviana fruit methanolic extract

Abstract Background: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. Methods: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. Results: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. Conclusion: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines. Keywords: Cytotoxic activity, Anti-proliferative activity, Motility, Apoptosis, Human cancer cells, Flavonoid, Cardiac glycoside

Catching a Ball at the Right Time and Place: Individual Factors Matter

Intercepting a moving object requires accurate spatio-temporal control. Several studies have investigated how the CNS copes with such a challenging task, focusing on the nature of the information used to extract target motion parameters and on the identification of general control strategies. In the present study we provide evidence that the right time and place of the collision is not univocally specified by the CNS for a given target motion; instead, different but equally successful solutions can be adopted by different subjects when task constraints are loose. We characterized arm kinematics of fourteen subjects and performed a detailed analysis on a subset of six subjects who showed comparable success rates when asked to catch a flying ball in three dimensional space. Balls were projected by an actuated launching apparatus in order to obtain different arrival flight time and height conditions. Inter-individual variability was observed in several kinematic parameters, such as wrist trajectory, wrist velocity profile, timing and spatial distribution of the impact point, upper limb posture, trunk motion, and submovement decomposition. Individual idiosyncratic behaviors were consistent across different ball flight time conditions and across two experimental sessions carried out at one year distance. These results highlight the importance of a systematic characterization of individual factors in the study of interceptive tasks