Global Medical Discovery features paper: Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Significance Statement

Arteries have the remarkable ability to adjust the amount and force of blood that flows through them. Such adjustments are made possible by contraction or relaxation of arterial smooth muscle cells (ASMCs) within the walls of the arteries. Many hormonal and neuronal actions serve to adjust the contraction of ASMCs, to modulate blood flow and pressure. At the cellular level, contraction of ASMCs depends on the flow of calcium ions into the cells through specialized protein pores or “channels” on the cell’s plasma membrane. Opening of voltage-sensitive calcium channels (VSCCs) involves positive changes to the voltage across the plasma membrane. Relaxation of ASMCs occurs when membrane voltage is maintained around -60 millivolts (negative inside compared to outside) by a flux of potassium ions out of the cells through channels that selectively conduct potassium; this negative voltage inhibits the opening of VSCCs. Among the many types of potassium channels on the ASMC plasma membrane, Kv7 channels, are particularly well suited as targets for hormonal and neuronal regulation of ASMC contraction to adjust blood pressure and blood flow.

Membrane voltage, and hence the flux of calcium ions via VSCCs, is very sensitive to the opening (activation) or closing (e.g. blocking) of Kv7 channels. These channels are tetrameric assemblies constituted by four Kv7 channel alpha subunits. There are five different types of Kv7 channel alpha subunits, named Kv7.1 through 7.5. In ASMCs, Kv7 channels are composed of four Kv7.4 subunits, four Kv7.5 subunits, or by some combination of Kv7.4/7.5 subunits (i.e. heterotetrameric channels). Altering the activity of the Kv7 channels with chemicals that bind directly to the channels has been shown to influence arterial contractility and diameter. However, physiological regulation of Kv7 channel activity is still poorly understood. In particular, it is unclear how the subunit composition of the channels influences the regulation of their activity.

In the article by Mani et al. (PMID: 26700561), the activity of ASMC Kv7 channels was monitored in response to activation of cell surface receptors and intracellular signaling regulators to better understand the physiological regulation of these channels. Furthermore, to study how different channel subunits respond to these stimuli, cultured rat aorta ASMCs (A7r5 cells) that naturally express only Kv7.5 subunits were used, and compared with the responses of ASMCs freshly isolated from rat arteries, which predominantly express heterotetrameric Kv7.4/Kv7.5 channels. In addition, human Kv7 channel alpha subunits were artificially introduced into A7r5 cells to compare regulation of human Kv7.4 or human Kv7.5 channels expressed individually or expressed together.

The results indicated that activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), a well-known vasodilatory stimulus, robustly increased the activity of Kv7.5 channels. Activation of cAMP/PKA using chemicals, or by engagement of beta adrenergic receptors that activate this signaling pathway, similarly increased the activity of naturally occurring Kv7.5 channels or of artificially introduced human Kv7.5 channels in A7r5 cells. In contrast, activity of freshly isolated rat artery Kv7.4/Kv7.5 channels or artificially introduced human Kv7.4/7.5 channels were only modestly enhanced, and human Kv7.4 channels were insensitive to activation of this signaling pathway. It was further demonstrated that the changes in activity of the channels by the signaling pathway are dependent on temporary addition of a phosphate group to the Kv7.5 channel subunits. No phosphate additions were detectable by activation of the signaling pathway in cells with artificially introduced Kv7.4 channels.

In summary, these results suggest that the responsiveness of arterial smooth muscle Kv7 channel subunits to intracellular cAMP/PKA signal activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. The differences in Kv7 channel subunit response may have important implications in terms of arterial function, as the Kv7 channel subunit expression patterns may differ among vascular beds and may change during development or with disease.

      

Figure Legend: Phosphorylation of Kv7.5 alpha subunits enhances efflux of potassium ions from arterial smooth muscle cells. Schematic diagram illustrating the signal transduction pathway whereby activation of β-adrenergic receptors (β-Adr) leads to elevation of cytosolic concentrations of cyclic adenosine monophosphate (cAMP) and hence to activation of Protein Kinase A. Protein kinase A can catalyze the transfer of phosphate (P) to Kv7.5 alpha subunits, which increases the opening of channels containing these subunits (Kv7.5 homotetramers or Kv7.4/Kv7.5 heterotetramers; solid arrows). Opening of the channels increases efflux of potassium ions (K+), which promotes smooth muscle relaxation. Kv7.4 alpha subunits are not phosphorylated by Protein Kinase A, and the activity of Kv7.4 homotetramers is not altered by this mechanism (dashed arrow).

 

About The Author

Bharath Mani received his DVM degree in 2001 and MS degree in 2003 from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, and Ph.D (Pharmacology) degree in 2012 from Loyola University Chicago, USA. His doctoral research examined the role of arterial smooth muscle Kv7 channel function in influencing arterial contraction status.

He is now a postdoctoral fellow at the University of Texas Southwestern Medical Center at Dallas, Texas, USA. His current research investigates neuroendocrine regulation of metabolic and cardiovascular function.

Journal Reference

Mol Pharmacol. 2016; 89(3):323-34.

Mani BK, Robakowski C, Brueggemann LI, Cribbs LL, Tripathi A, Majetschak M, and Byron KL.

Loyola University Chicago, Dept. of Molecular Pharmacology & Therapeutics, Maywood, IL 60153, USA.

Abstract

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth musclecell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth musclecells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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Global Medical Discovery features paper: Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions

Journal Reference

Biochem Pharmacol. 2016;103:129-39.

Saenz-de-Viteri M¹, Fernández-Robredo P², Hernández M³, Bezunartea J⁴, Reiter N⁴, Recalde S², García-Layana A⁵.

Show Affiliations

1. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain.
2. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
3. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain. Electronic address: mahersan@unav.es.
4. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain.
5. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.

 

Abstract

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under  normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under  normal and  oxidative  stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5days after treatment. Finally, none of the drugs affected migration.

In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Global Medical Discovery features paper: Resveratrol induces ordered domains formation in biomembranes: Implication for its pleiotropic action

Significance Statement

Membrane Approach to Understand the Pleiotropic Effects of Resveratrol

Resveratrol is a natural polyphenol compound with great potential in the cancer therapy, cardiovascular protection, and neurodegenerative disorders. The mechanism of action of resveratrol may be associated to its capacity of modulating cell membrane structure and function, thereby influencing the activity of several membrane associating receptors, proteins and enzymes. The present research reveals potential molecular interactions between resveratrol and lipid rafts found in cell membranes, bringing a new membrane approach to understand the pleiotropic effects of this phytochemical.

Förster resonance energy transfer (FRET), DPH fluorescence quenching by TEMPO, and Triton X-100 detergent resistance assay have been employed to monitor lipid phase separation in model membranes. Unilamellar liposomes composed of phosphatidylcholine, cholesterol and sphingomyelin were chosen as membrane mimetic systems. The results indicate that resveratrol is able to incorporate in lipid bilayers, inducing phase separation, stabilizing and promoting the formation of ordered domains (lipid rafts) that can act as organizing centers for the assembly of proteins and receptors, which in turn may be involved in the cell signaling and cellular processes.

In conclusion, the influence of resveratrol on the regulation of lipid domains formation and stability provide a rational approach to better understand the therapeutic effects of this promising compound.

About The Author

Dr. Ana Rute Neves (PhD Pharmaceutical Sciences in 2015) is a Post-doctoral researcher in the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her research focuses on biophysical and biochemical approaches to study the interaction of phytochemicals with biomembranes (using liposomes as biomimetic systems) in order to explain their therapeutic properties and the development of nanopharmaceuticals (lipid nanoparticles) as drug delivery systems to create new and more efficient therapies for a range of diseases.

About The Author

Dr. Cláudia Nunes (PhD Pharmaceutical Sciences in 2011) is a Post-doctoral researcher in the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her research activities include the assessment of drug-membrane interactions, using membrane mimetic models; the development of nanotechnology based drug delivery systems (silica nanotubes, solid lipid nanoparticles, nanostructured lipid particles and liposomes) that can be efficiently carried to the inflamed tissues avoiding the gastric local toxic effects of the classical therapies.

About The Author

Professor Salette Reis (PhD Analytical Chemistry in 1995) is the Director of the Department of Chemical Sciences and the group leader of the Molecular Biophysics and Biotechnology Unit at GABAI/UCIBIO/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal. Her investigation has been focused in biophysics and pharmaceutical chemistry, namely in the use of biomimetic membrane models to study the effect of drugs on biological membranes trying to establish a relationship between this effect and their activity/mechanism of action; and the development of nanocarrier systems for drug delivery to overcome the disadvantages of the classical therapies.

Journal Reference

Biochim Biophys Acta. 2016;1858(1):12-8.  

Neves AR¹, Nunes C¹, Reis S².

Show Affiliations

1. UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
2. UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: shreis@ff.up.pt.

Abstract

Resveratrol is a polyphenol compound with great value in cancer therapy, cardiovascular protection, and neurodegenerative disorders. The mechanism by which resveratrol exerts such pleiotropic effects is not yet clear and there is a huge need to understand the influence of this compound on the regulation of lipid domains formation on membrane structure. The aim of the present study was to reveal potential molecular interactions between resveratrol and lipid rafts found in cell membranes by means of Förster resonance energy transfer, DPH fluorescence quenching, and triton X-100 detergent resistance assay. Liposomes composed of egg phosphatidylcholine, cholesterol, and sphingomyelin were used as model membranes. The results revealed that resveratrol induces phase separation and formation of liquid-ordered domains in bilayer structures. The formation of such tightly packed lipid rafts is important for different signal transduction pathways, through the regulation of membrane-associating proteins, that can justify several pharmacological activities of this compound.

Copyright © 2015 Elsevier B.V. All rights reserved.

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Global Medical Discovery features paper: An integrated hybrid microfluidic device for oviposition-based chemical screening of adult Drosophila melanogaster

Significance Statement

Model organisms play an important role in the cycle of drug discovery. Drosophila melanogaster is one of the invertebrate model organisms that has been extensively used to study human diseases and disorders. However, the widespread use of this organism has been impeded by the lack of automated and high throughput technologies for screening. In this article, we have developed a microfluidic device (shown schematically) consisting of porous agar with integrated networks of microchannels used to selectively infuse chemicals at desired concentrations to favorable locations in the device. We have used this technology to study the effect of zinc and acetic acid on oviposition or egg laying behavior of adult fruit flies. Our findings show that fruit flies are capable of sensing the concentration of chemicals in single-chemical assays and use this functionality to explore and select desirable oviposition sites in multi-chemical platforms. This technology can be used for screening drugs, studying learning and memory functions, and investigating biological pathways of oviposition in agricultural, drug discovery, biological, and medical applications.  

About The Author

Jacob C.K. Leung received his Bachelor of Engineering degree with honors distinction in Biomedical Engineering from Ryerson University, Toronto, ON, Canada. He obtained his Master of Science degree in Mechanical Engineering at York University under the supervision of Professor Pouya Rezai. His research focused on developing efficient microfluidic devices to perform toxicological assays on Drosophila melanogaster. He also gained expertise in developing sacrificial layer based microfabrication techniques to produce PDMS and composite PDMS micropillars and microbridges for sensor applications. In addition to his engineering background, Jacob is also pursuing higher education in Occupational Therapy at the University of Toronto. Integrating his engineering skills with rehabilitation medicine, Jacob’s research interests include developing person-centered assistive devices and automated, low-cost, and high-throughput biomedical diagnostic technologies for developmental biology, toxicology, and drug discovery. 

About The Author

Professor Arthur Hilliker completed his PhD at the University of British Columbia (1975) and pursued postdoctoral work at the University of Connecticut before joining CSIRO in Australia as a research scientist (1978) in the Division of Plant Industry. His first academic appointment was at University of Guelph in 1982 as an Assistant Professor, advancing through the ranks to Professor in 1993. Dr. Hilliker moved to York University to be the Chair of the Department of Biology in 2000. Dr. Hilliker has had a long standing record of service to the scientific community. He was the first person awarded the Young Scientist Award from the Genetics Society of Canada (1987). In 2005, he was again recognized by the Genetics Society of Canada with the Award of Excellence for lifetime scientific contributions to genetics. He was Co-Editor in Chief of the National Research Council of Canada Press journal Genome from 2007 until 2014. Since 2010 he has served on the Executive of the Canadian Society of Biochemistry and Molecular and Cell Biology (now renamed the Canadian Society of Molecular Biosciences) and is currently a Past President and Treasurer. His research is primarily although not entirely focused on using Drosophila as a model organism. His research covers a wide breadth of subject areas relating to genetics. 

About The Author

Professor Pouya Rezai is an emerging researcher in the area of microfluidics and Lab-on-a-Chip (LoC) devices. He is an Assistant Professor and the Graduate Program Director at the Department of Mechanical Engineering at York University. He received his Master of Science in Electrical Engineering from Chalmers University of Technology in 2008 and his PhD in Mechanical Engineering from McMaster University in 2012. Dr. Rezai was an NSERC Visiting Fellow at Public Health Agency of Canada before joining York University in July 2013. His research interest includes advancing micromachining and microfabrication techniques to develop LoC devices to study interactions between micro-particles, small biological substances and fluids in microenvironments. He has developed (i) LoC devices for quantitative investigation of neurobehavioral responses of bio-organisms (e.g., D. melanogaster, C. elegans, D. rerio) to electric, acoustic, or chemical cues; (ii) microfluidic platforms for multiplexed sorting of pathogens and microparticles in fluidic samples; and (iii) micro-electro-mechanical sensors for measuring properties of polymers and carbon nano-structures.

Journal Reference

Lab Chip. 2016 Feb 21;16(4):709-19.

Leung JC, Hilliker AJ, Rezai P.

Department of Mechanical Engineering, York University, BCEE 433B, 4700 Keele St, Toronto, ON M3J 1P3, Canada. prezai@yorku.ca.

Abstract

Chemical screening using Drosophila melanogaster (the fruit fly) is vital in drug discovery, agricultural, and toxicological applications. Oviposition (egg laying) on chemically-doped agar plates is an important read-out metric used to quantitatively assess the biological fitness and behavioral responses of Drosophila. Current oviposition-based chemical screening studies are inaccurate, labor-intensive, time-consuming, and inflexible due to the manual chemical doping of agar. In this paper, we have developed a novel hybrid agar-polydimethylsiloxane (PDMS) microfluidic device for single- and multi-concentration chemical dosing and on-chip oviposition screening of free-flying adult stage Drosophila. To achieve this, we have devised a novel technique to integrate agar with PDMS channels using ice as a sacrificial layer. Subsequently, we have conducted single-chemical toxicity and multiple choice chemical preference assays on adult Drosophila melanogaster using zinc and acetic acid at various concentrations. Our device has enabled us to 1) demonstrate that Drosophila is capable of sensing the concentration of different chemicals on a PDMS-agar microfluidic device, which plays significant roles in determining oviposition site selection and 2) investigate whether oviposition preference differs between single- and multi-concentration chemical environments. This device may be used to study fundamental and applied biological questions in Drosophila and other egg laying insects. It can also be extended in design to develop sophisticated and dynamic chemical dosing and high-throughput screening platforms in the future that are not easily achievable with the existing oviposition screening techniques.

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Definition of an 18-mer Synthetic Peptide Derived from the GB virus C E1 Protein as a New HIV-1 Entry Inhibitor

Significance Statement

In this article we report the construction of several peptide libraries scanning the GB virus C (GBV-C) E1 protein and the evaluation of their anti-HIV-1 activity. On the basis of the results, an 18-mer synthetic peptide, specifically the sequence E1(139-156) (namely E1P47), is defined as a new anti-HIV-1 peptide entry inhibitor. The peptide showed similar antiviral activity against viruses from clades A, B, C, D and AE. The broad spectrum of activity against HIV-1 that E1P47 has been demonstrated to have, as well as its capacity to prevent HIV infection by inhibiting (at least partially) viral entry, constitute favourable properties when evaluating this small peptide as a microbicide lead. We consider that these results are of interest for people working in the field of discovering new peptides with the ability of inhibiting HIV and more specifically those involved in studying the relationship of GB virus C and HIV viruses. The results described in the present work indicate the implication of the E1 envelope of the GB virus C in the inhibition of HIV-1. Moreover, our results also highlight the definition of one inhibiting domain within this protein. Thus, taken together the results allow us to consider also the non-pathogenic E1 GB virus C protein as an attractive source of peptides for the development of novel anti-HIV therapies. We strongly believe that the results reported here are important in themselves, so far as we have defined a novel lead compound as in that for the first time the inhibitory role of a highly conserved fragment of the E1 protein is reported.

Group information

The research interests of the Unit of Synthesis and Biomedical Applications of Peptides deal with the use of peptides for the development of new biosensors for the diagnosis of human illnesses and on the selection of therapeutic agents of peptide origin through biophysical testing.

Specifically, in the field of synthetic peptides for the diagnosis and prognosis of rheumatoid arthritis, we aim to identify new antigen peptides, derived from citrullinated/homocitrullinated proteins present in rheumatoid synovial fluid (fibrin, filaggrin, vimentin and enolase) in order to thereby identify those patients who require more aggressive therapies right from the moment of diagnosis of the disease. That would allow greater control of the disease and, consequently, less harm to the articulations and a better prognosis. Moreover, the detection of a single biomarker is not always sufficiently sensitive or precise to discriminate between RA patients with different prognoses, clinical characteristics or responses to therapy. Through the simultaneous analysis of the target peptides, incorporated into a multiplex test, we aim to facilitate the biological fingerprinting of autoantibodies in serum that will be able to identify subgroups of patients with specific clinical characteristics; different prognoses; and who either respond well to, or suffer negative effects from, certain therapeutic interventions. These results could have far-reaching practical implications for establishing the most appropriate therapeutic strategy for RA patients.

Related with the study of peptide human immunodeficiency virus entry inhibitors, we propose the study of new therapeutic agents for the treatment of AIDS, together with the development of a biosensor platform that can be used to detect antibodies against GB virus C in patients co-infected with HIV-1.

Finally, the objective of the research line on peptide controlled-release nanosystems for ocular administration of drugs includes the development of new systems of administration based on liposomes and nanoparticles that are targeted via the use of peptides and that can ensure low levels of irritation, sufficient bioavailability and compatibility with ocular tissues.

About The Author

Prof. Isabel Haro. Senior Staff. B.Sc. Chemistry, University of Barcelona, 1982. M.Sc. Biochemistry,University of Barcelona, 1984. Ph.D. Chemistry, University of Barcelona, 1988. Postdoctoral fellow (1989-1990) between CSIC in Barcelona and Royal Free Hospital in London (Prof. G. Gregoriadis). Research Scientist at CSIC leading the Unit of Synthesis and Biomedical Applications of CSIC in 1990. Since then she has been working in the use of synthetic peptides in the field of biomedicine, specifically in the development of new biosensors for the diagnosis of human illnesses and in the selection of new therapeutic agents of peptide origin through biophysical testing. She has published more than 180 articles, 8 reviews, 30 chapters of books and 8 patents. Also, she has been the principal researcher of 15 research projects and 3 contracts with the industry and supervised 12 doctoral theses. e-mail contact: isabel.haro@iqac.csic.es  

About The Author

Dr. Maria JoséGomara. Senior Staff. B.Sc. Pharmacy, University of Barcelona, 1992. M.Sc. Physical Chemistry, University of Barcelona, 1995. Ph.D. Pharmacy, University of Barcelona, 2000. Postdoctoral fellow (2001-2004) in Prof. Felix Goñi laboratory (Biophysical Unit, CSICEHU/UPV). Research assistant (2005-2007) in the Unit of Synthesis and Biomedical Applications of Peptides leaded by Dr. Haro, IQAC-CSIC. Research scientist of CSIC since 2008. Experience in peptide synthesis, biophysical studies of peptide-lipid and peptide-peptide interactions and development of peptide-based immunoassays. She has published over 60 articles and 5 patents. e-mail contact: mariajose.gomara@iqac.csic.es 

Journal Reference

Biochim Biophys Acta. 2016 Feb 22;1860(6):1139-1148.

Gómara MJ¹, Sánchez-Merino V², Paús A³, Merino-Mansilla A², Gatell JM⁴, Yuste E², Haro I⁵.

Show Affiliations

1. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. Electronic address: mariajose.gomara@iqac.csic.es.
2. AIDS Research Unit, Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain. HIVACAT, Barcelona, Spain.
3. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain.
4. AIDS Research Unit, Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain. HIVACAT, Barcelona, Spain; Infectious Diseases Unit-HIVACAT, Hospital Clinic, Villarroel, 170, 08036 Barcelona, Spain.
5. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. Electronic address: isabel.haro@iqac.csic.es.

Abstract

BACKGROUND:

A slower progression of AIDS and increased survival in GB virus C positive individuals, compared with GB virus C negative individuals has been demonstrated; while the loss of GB virus C viremia was closely associated with a rise in mortality and increased progression of AIDS. Following on from the previous reported studies that support the thesis that GB virus C E2 interferes with HIV-1 entry, in this work we try to determine the role of the GB virus C E1 protein in HIV-1 inhibition.

METHODS:

The present work involves the construction of several overlapping peptide libraries scanning the GBV-C E1 protein and the evaluation of their anti-HIV activity.

RESULTS:

Specifically, an 18-mer synthetic peptide from the GB virus C E1 protein, E1(139-156), showed similar antiviral activity against HIVs from viruses from clades A, B, C, D and AE. Competitive ELISA using specific gp41-targeting mAbs, fluorescence resonance energy transfer as well as haemolysis assays demonstrated that this E1 peptide sequence interacts with the highly conserved N-terminal region of the HIV-1 gp41 (the fusionpeptide) which is essential for viral entry.

CONCLUSIONS:

We have defined a novel peptide lead compound and described the inhibitory role of a highly conserved fragment of the E1 protein.

GENERAL SIGNIFICANCE:

The results together allow us to consider the non-pathogenic E1 GB virus C protein as an attractive source of peptides for the development of novel anti-HIV therapies.

Copyright © 2016 Elsevier B.V. All rights reserved.

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Cerebral mast cells contribute to postoperative cognitive dysfunction by promoting blood brain barrier disruption

About The Author

Yanning Qian, Director of Department of Anesthesiology Research, First affiliated hospital of Nanjing Medical University, Deputy Director of the General Surgery Department of Nanjing Medical University. Research interests: anesthetic effects on nerve-endocrine-immune network. This research direction have had some effect in the domestic and foreign counterparts: achieved the provincial science and technology progress third class Prize 2 times; achieved 3 National Natural Science Foundation; in 2011, achieved one of the Ministry of education doctoral project; published more than 20 papers in SCI journals as first or corresponding author; several papers presented in international conferences. 

Journal Reference

Behav Brain Res. 2016 Feb 1;298(Pt B):158-66.

Zhang S¹, Dong H¹, Zhang X¹, Li N¹, Sun J², Qian Y³.

Show Affiliations

1. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China.
2. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China. Electronic address: dgsunjie@hotmail.com.
3. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China. Electronic address: yanning_qian@163.com.

Abstract

Trauma induced neuroinflammation plays a key role in the development of  postoperative  cognitive dysfunction  . The blood-brain barrier (BBB), a highly specialized endothelial layer, is exquisitely sensitive to inflammatory insults, which can result in numerous neurocognitive syndromes. While brain mast cells are the “first responder” in the injury, the functional interactions between mast cells and the BBB remain poorly understood. Our results demonstrate that tibial fracture surgery can induce cognitive impairment relating to an inflammatory response and destabilization of the BBB. Disodium cromoglycate (cromolyn) – which acts as a mast cell stabilizer – inhibited this effect. Specifically, cromolyn resulted in ameliorated cognitive ability, decrease of inflammatory cytokines and increase of BBB stability. Taken together, these results suggest that activated mast cells contributed to central nervous system inflammation and cognitive dysfunction by promoting BBB disruption, and interactions between mast cells and the BBB could constitute a new and unique therapeutic target for postoperative cognitive dysfunction.

Copyright © 2015 Elsevier B.V. All rights reserved.

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Role of iso-receptors in receptor-receptor interactions with a focus on dopamine iso-receptor complexes

Significance Statement

The discovery of receptor-receptor interactions (RRIs) in the early 1980s together with a more accurate focusing of allosteric mechanisms in proteins, significantly expanded the knowledge on the G protein-coupled receptors (GPCR)-mediated signaling processes in the cells. In particular, increasing evidence was provided that GPCR operate not only as monomers but also as quaternary structures (homodimers, heterodimers and higher-order oligomers), in which networks of electrostatic interactions (hydrogen bonds, van der Waals forces) shape the configuration of the single receptors and the topology of the entire complex, allowing an integration of the incoming signals already at the plasma membrane level through allosteric receptor-receptor interactions. Once established, these integrative mechanisms change the function of the involved GPCR, leading to a sophisticated dynamics of the receptor assembly in terms of modulation of recognition and signaling.

As a consequence, when the interacting receptors are iso-receptors a single neurotransmitter becomes able to induce a spectrum of possible cellular responses that can either work in parallel (redundancy) or interact with each other (complex responses). These factors lead to an astonishing increase in the modes for the recognition/decoding processes that control the cellular biochemical machineries and appear of particular interest for possible therapeutic applications, as indicated by studies on the dopamine iso-receptor complexes D1-D2 and D1-D3 suggesting them as possible targets for neuropsychiatric disorders.

Thus, the expansion of the GPCR field emerging from the characterization of receptor-receptor interactions can also pave the way for the development of novel pharmacological strategies for the treatment of several pathologies, and in the next future this research effort is likely to have a major impact on molecular medicine.

Figure Legend: Micro-domains of the plasma membrane where Receptor-Receptor Interactions (RRIs) can lead to the formation of receptor complexes (Receptor Mosaics, RMs).  From the topology and dynamics of these receptor assemblies a wide spectrum of possible cellular responses to an incoming signal can emerge (Agnati, Guidolin et al., Prog Neurobiol, 2010)

Journal Reference

Rev Neurosci. 2016 Jan 1;27(1):1-25.

Luigi F. Agnati¹ , Diego Guidolin² , Chiara Cervetto³ , Dasiel O. Borroto-Escuela⁴ , Kjell Fuxe⁴ 

Show Affiliations

1. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Via Campi 287, 41100 Modena, Italy
2. Department of Molecular Medicine, University of Padova, Via Gabelli 65, 35121 Padova, Italy
3. Department of Pharmacy, University of Genova, Viale Cembrano 4, 16147 Genova, Italy
4. Department of Neuroscience, Karolinska Institutet, Retzius vag 8, 17177 Stockholm, Sweden

Abstract

Intercellular and intracellular communication processes consist of signals and recognition/decoding apparatuses of these signals. In humans, the G protein-coupled receptor (GPCR) family represents the largest family of cell surface receptors. More than 30 years ago, it has been proposed that GPCR could form dimers or higher-order oligomers (receptor mosaics [RMs] at the plasma membrane level and receptor-receptor interactions [RRIs] have been proposed as a new integrative mechanism for chemical signals impinging on cell plasma membranes). The basic phenomena involved in receptor-receptor interactions  are allostery and cooperativity of membrane receptors, and the present paper provides basic information concerning their relevance for the integrative functions of RMs. In this context, the possible role of iso-receptor RM is discussed (with a special focus on dopamine receptor subtypes and on some of the RMs they form with other dopamine iso-receptors), and it is proposed that two types of cooperativity, namely, homotropic and heterotropic cooperativity, could allow distinguishing two types of functionally different RMs. From a general point of view, the presence of iso-receptors and their topological organization within RMs allow the use of a reduced number of signals for the intercellular communication processes, since the target cells can recognize and decode the same signal in different ways. This theoretical aspect is further analyzed here by means of an analogy with artificial information systems. Thus, it is suggested that the ‘multiplexer’ and ‘demultiplexer’ concepts could, at least in part, model the role of RMs formed by iso-receptors in the information handling by the cell.

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Thymidine 5′-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor

Significance Statement

Ligands of the P2Y nucleotide receptors are of potential interest for pharmacotherapies directed at neurodegenerative disorders, intestinal function, angiogenesis, cardiac remodeling,  etc.  Activation of P2Y6 by its native ligand UDP or by synthetic agonists is associated with vasoconstriction and cytoprotection. P2Y6 seems to control chloride secretion by epithelium, the direct contraction and endothelium-dependent relaxation of the aorta or insulin secretion as well. Pharmacological modulation of the P2Y6 receptor is of increasing interest, nevertheless UDP as a natural ligand was reported to activate not only P2Y6 but also P2Y14. Therefore, agonists that delineate between these two subtypes are needed. Besides, the therapeutic potential of extracellular nucleotides is limited because they are degraded by extracellular enzymes which reduce their efficacy and duration of action. We pay particular attention to the unique activity of thymidine 5’-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor. Its  increased stability as compared to their unmodified counterpart as well as their affinity towards P2Y6 is especially interesting and may be responsible for some long-term effects.  

About The Author

Edyta Gendaszewska-Darmach:

PhD degree in Bioorganic Chemistry from Polish Academy of Sciences.

an Asissstant Professor at Faculty of Biotechnology and Food Sciences (Institute of Technical Biochemistry),  Lodz University of Technology.

Research involves anti-diabetic and wound healing properties of natural and modified biophosphates (nucleotides and lysophospholipids) as well as biomaterials for regenerative medicine.

Journal Reference

Purinergic Signal. 2016 Jan 8.

Gendaszewska-Darmach E¹, Szustak M².

Show Affiliations

1. Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924, Lodz, Poland. edarmach@wp.pl.
2. Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924, Lodz, Poland.

Abstract

ATP, ADP, UTP, and UDP acting as ligands of specific P2Y receptors activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, migration, differentiation, and cell death. Contrary to a widely held opinion, we show here that nucleoside 5′-O-monophosphorothioate analogs, containing a sulfur atom in a place of one nonbridging oxygen atom in a phosphate group, act as ligands for selected P2Y subtypes. We pay particular attention to the unique activity of thymidine 5′-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor (P2Y6R). We also collected evidence for the involvement of the P2Y6 receptor in human epithelial adenocarcinoma cell line (HeLa) cell migration induced by thymidine 5′-O-  monophosphorothioate analog. The stimulatory effect of TMPS was abolished by siRNA-mediated P2Y6 knockdown and diisothiocyanate derivative MRS 2578, a selective antagonist of the P2Y6R. Our results indicate for the first time that increased stability of thymidine 5′-O-monophosphorothioate as well as its affinity toward the P2Y6R may be responsible for some long-term effects mediated by this receptor.

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A liposomal method for evaluation of inhibitors of H(+)-coupled multidrug transporters

Journal Reference

J Pharmacol Toxicol Methods. 2016 Jan-Feb;77:53-7.

Melchior DL¹, Brill S², Wright GE³, Schuldiner S².

Show Affiliations

1. GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA. Electronic address: donald.melchior@glsynthesis.com.
2. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, Hebrew University, Jerusalem 91904, Israel.
3. GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA.

Abstract

INTRODUCTION:

This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance.

METHODS:

To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized.

RESULTS:

Fluorosomes containing the Escherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC50 for nigericin inhibition of ethidium was shown to be 0.71μM.

DISCUSSION:

We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome-trans-EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Suppression of Th2 and Tfh immune reactions by Nr4a receptors in mature T reg cells

Significance Statement

Immune systems protect our body from threats by various infectious pathogens, including bacteria, virus, helminth, and fungi. In the immune systems, CD4⁺ helper T cells (Th) play central roles to exert appropriate immune reactions to such innumerable sorts of pathogens. However, if the function of Th are wrongly directed to self-antigens or non-harmful antigens, including pollens, food antigens, and commensals, it will be concluded to serious inflammatory diseases like autoimmune diseases and allergies. On the other hand, immune system has an important cell subset, regulatory T (Treg) cells, which suppress such wrong immune reactions. Treg cells have been known to be critical in suppression of inflammatory diseases, however, how Treg cell lineage was properly maintained in our body had not been clarified in depth yet. In this study, we discovered that Nr4a family of nuclear orphan receptors play critical roles in the maintenance of the Treg cell lineage. Nr4a-deficient Treg cells showed a global reduction of Treg cell-associated gene expressions, compared with those in wildtype Treg cells. Although wildtype Treg cells properly suppressed immune reactions elicited by Th2 and Tfh cells, it was revealed that Nr4a-deficient Treg cells have lost such suppressive activities. We also found that Nr4a-deficient Treg cells aberrantly convert to cells which have characteristics of inflammatory Th cells. Mice in which Nr4a factors were deleted specifically in Treg cells develop symptoms including allergic asthma and autoimmune diseases. Collectively, our work revealed that Nr4a factors play essential roles in Treg cell biology, thus present attractive therapeutic targets for treatment of various immune disorders, including atopic allergies and asthma. 

About The Author

Dr. Takashi Sekiya received his Bachelor of Science degree in 1999 from University of Tokyo. He obtained his Ph.D. from Graduate School of Agricultural and Life Sciences, The University of Tokyo in 2004. Then, he moved to Fox Chase Cancer Center as a postdoctoral fellow at Dr. Ken Zaret’s laboratory. In 2008, he moved back to Japan, and joined Dr. Akihiko Yoshimura’s lab in Keio University School of Medicine, there he has been working on mechanisms of immune tolerance, focusing on developmental programs of CD4 T cells. 

Journal Reference

J Exp Med. 2015;212(10):1623-40.

Sekiya T¹, Kondo T², Shichita T², Morita R², Ichinose H³, Yoshimura A⁴.

Show Affiliations

1. Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan yoshimura@a6.keio.jpt-sekiya@z7.keio.jp.
2. Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan.
3. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501, Japan.
4. Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo 102-0075, Japan yoshimura@a6.keio.jpt-sekiya@z7.keio.jp.

Abstract

Regulatory T (T reg) cells are central mediators of immune suppression. As such, T reg cells are characterized by a distinct pattern of gene expression, which includes up-regulation of immunosuppressive genes and silencing of inflammatory cytokine genes. Although an increasing number of transcription factors that regulate T reg cells have been identified, the mechanisms by which the T reg cell-specific transcriptional program is maintained and executed remain largely unknown. The Nr4a family of nuclear orphan receptors, which we recently identified as essential for the development of T reg cells, is highly expressed in mature T reg cells as well, suggesting that Nr4a factors play important roles even beyond T reg cell development. Here, we showed that deletion of Nr4a genes specifically in T reg cells caused fatal systemic immunopathology. Nr4a-deficient Treg cells exhibited global alteration of the expression of genes which specify the T reg cell lineage, including reduction of Foxp3 and Ikzf4. Furthermore, Nr4a deficiency abrogated T reg cell suppressive activities and accelerated conversion to cells with Th2 and follicular helper T (Tfh) effector-like characteristics, with heightened expression of Th2 and Tfh cytokine genes. These findings demonstrate that Nr4a factors play crucial roles in mature T reg cells by directly controlling a genetic program indispensable for T reg cell maintenance and function.

© 2015 Sekiya et al.

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Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites

Journal Reference

Mol Pharmacol. 2015 Dec;88(6):970-81.

Ozer U¹, Barbour KW¹, Clinton SA¹, Berger FG².

Show Affiliations

1. Department of Biological Sciences, and Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina.
2. Department of Biological Sciences, and Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina fgberger@mailbox.sc.edu.

Abstract

Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming dTMP for the maintenance of DNA replication and repair. Inhibitors of Thymidylate synthase have been widely used in the treatment of neoplastic disease. A number of fluoropyrimidine and folate analogs have been developed that lead to inhibition of the enzyme, resulting in dTMP deficiency and cell death. In the current study, we have examined the role of oxidative stress in response to TYMS inhibitors. We observed that intracellular reactive oxygen species (ROS) concentrations are induced by these inhibitors and promote apoptosis. Activation of the enzyme NADPH oxidase (NOX), which catalyzes one-electron reduction of O2 to generate superoxide (O2 (●-)), is a significant source of increased ROS levels in drug-treated cells. However, gene expression profiling revealed a number of other redox-related genes that may contribute to ROS generation. Thymidylate synthase inhibitors also induce a protective response, including activation of the transcription factor nuclear factor E2-related factor 2 (NRF2), a critical mediator of defense against oxidative and electrophilic stress. Our results show that exposure to Thymidylate synthase inhibitors induces oxidative stress that leads to cell death, while simultaneously generating a protective response that may underlie resistance against such death.

Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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Oncolytic adenovirus targeting cyclin E overexpression repressed tumor growth in syngeneic immunocompetent mice

Significance Statement

Induction of cyclin E by adenoviral E1B55K protein is required for productive virus replication in normal cells. However this viral protein is not required in cancer cells, allowing E1B55K-deleted adenoviruses to conduct oncolytic replication (Fig. A). In many cancer cells cyclin E expression is already deregulated, likely because that the cellular factors targeted by E1B55K may already be activated or there are E1B55K-like factors to relax cyclin E regulation. As the cyclin E promoter is highly active in many types of cancer cells and is further stimulated after adenovirus infection, Cheng and colleagues first applied this unique property of cyclin E promoter into the design of a novel tumor-specific oncolytic adenoviral vector Ad-cycE, in which the cyclin E promoter was used to control the critical regulatory viral E1a gene. Significant and selective antitumor efficacy can be achieved with Ad-cycE (Fig. B). In this study, Cheng et al. further present an interesting new model for the study in an immunocompetent preclinical model of lung cancer. This new model could be of great value in the preclinical development of the new anticancer agents and allow researchers to better evaluate oncolytic adenoviral vectors in a more patient-like setting.

About The Author

Dr. Pei-Hsin Cheng received her Ph.D. degree in Pharmacology and Toxicology from the University of Louisville, in 2013. With the motivation to find innovative biotherapeutics and to develop rational combination strategies for cancer treatments, she joined the St. Jude Children’s Research Hospital as a Postdoctoral Research Associate since 2014. Her research interests include oncolytic virotherapy, gene therapy, miRNA replacement therapy and immunotherapy.

Journal Reference

BMC Cancer. 2015;15:716.

Cheng PH¹, Rao XM², Wechman SL^3,4, Li XF⁵, McMasters KM^6,7, Zhou HS^8,9,10. 

Show Affiliations

1. Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, 40292, USA. paisin.paisin@gmail.com.
2. James Graham Brown Cancer Center, University of Louisville Medical School, 505 South Hancock Street, CTR Building, Room 306, Louisville, KY, 40202, USA. x0rao001@louisville.edu.
3. Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, 40292, USA. slwech01@exchange.louisville.edu.
4. Hiram C. Polk Jr MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY, 40292, USA. slwech01@exchange.louisville.edu.
5. Department of Diagnostic Radiology, University of Louisville School of Medicine, Louisville, KY, 40292, USA. x0li0027@exchange.louisville.edu.
6. Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, 40292, USA. mcmasters@louisville.edu.
7. Hiram C. Polk Jr MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY, 40292, USA. mcmasters@louisville.edu.
8. James Graham Brown Cancer Center, University of Louisville Medical School, 505 South Hancock Street, CTR Building, Room 306, Louisville, KY, 40202, USA. hszhou01@louisville.edu.
9. Hiram C. Polk Jr MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY, 40292, USA. hszhou01@louisville.edu.
10. ⁰Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, 40292, USA. hszhou01@louisville.edu.

Abstract

BACKGROUND:

Clinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads.

METHODS:

Ad-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies.

RESULTS:

Murine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment.

CONCLUSION:

Ad-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor.

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Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network

Significance Statement

In the manuscript “Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network” a combination therapy comprising chemotherapeutics and death receptor agonists was analyzed in an in vitro model of pancreatic cancer.  This co-treatment led to a strong synergistic effect, killing a high amount of cancer cells. More detailed analysis revealed a sensitization mechanism via down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, which are typically overexpressed in cancer cells thereby causing defects in the cell death machinery. This gemcitabine-mediated decrease of c-FLIP protein levels changed the composition of the death-inducing signaling complex (DISC) after CD95L-treatment towards a higher amount of the apoptosis-inducing proteins Caspase-8 and Caspase-10. This modified protein complex composition is linked to the higher rate of cell death induction. Cell death assays in presence of inhibitors of apoptosis (zVAD-fmk) and necroptosis (Necrostatin-1) indicated that the combination of gemcitabine and CD95L induced both cell death modes, apoptosis and necroptosis, while the stimulation with CD95L alone led solely to apoptosis. Additionally, the usage of the cutting edge technology “imaging flow cytometry” confirmed that the combinatorial treatment not only sensitized cells to death, but also switched them to an alternative cell death pathway – necroptosis via RIPK1. These results demonstrate that this cell death network is regulated via caspase-dependent and caspase-independent pathways. The combined therapy of chemotherapeutics and death receptor agonists might offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-mediated cell death.  These findings might improve treatment strategies and are important for the development of the systems biology-based approaches for the personalized medicine.

 

About The Author

Dr. Sabine Pietkiewicz studied Biology at the Heinrich-Heine-University in Düsseldorf, Germany and already started focusing on apoptosis and necroptosis after death receptor stimulation during her diploma thesis in the Institute of Molecular Medicine, under the supervision of Prof. Schulze-Osthoff. In Düsseldorf she obtained her Dr. rer. nat. at the Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, and has characterized the role of JNK isoforms in cell death pathways after treatment with the proteasomal inhibitor and chemotherapeutic Bortezomib. She currently conducts research in the department Translational Inflammation Research of Prof. Inna N. Lavrik at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany, analyzing the CD95/APO-1/Fas signaling networks.  

About The Author

Prof. Inna N. Lavrik is a head of department Translational Inflammation Research at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany. She did her Postdoctoral training in Heidelberg, at German Cancer Research Center, at the Division of Immunogenetics, Head Prof. Dr. Peter H. Krammer.  Afterwards she had a group leader position at DKFZ/Bioquant, Heidelberg, Germany, which she held before moving to Magdeburg.

Journal Reference

Exp Cell Res. 2015;339(1):1-9.

Pietkiewicz S¹, Eils R², Krammer PH³, Giese N⁴, Lavrik IN⁵. 

Show Affiliations

1. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany.
2. Bioquant, Heidelberg University, 69120 Heidelberg, Germany; Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany; Division of Theoretical Bioinformatics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
3. Division of Immunogenetics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
4. Department of General Surgery, University of Heidelberg, Germany (g)Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia.
5. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany; Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia. Electronic address: inna.lavrik@med.ovgu.de.

Abstract

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Design and synthesis of 2-phenylnaphthalenoids and 2-phenylbenzofuranoids as DNA topoisomerase inhibitors and antitumor agents

Significance Statement

Human DNA topoisomerase IIα (TopoIIα), an Achilles’ heel of cancer, is a well-validated target for antitumor drugs. The inhibitors of TopoIIα are among the most effective and commonly used antitumor drugs in clinic, e. g. etoposide and doxorubicin. However, clinically used v inhibitors exhibited serious side effects including cardiotoxicity, development of secondary malignancies and multidrug resistance, thus that it is urgent to explore novel DNA topoisomerase IIα inhibitors for the development of antitumor drugs.

In the recent work of Shen laboratory, a group of 2-phenylnaphthalenoids (2PNs) and 2-phenylbenzofuranoids (2PBFs) were designed and synthesized for the sake of innovation of DNA topoisomerase IIα inhibitors. These 2PNs and 2PBFs were proved to display favorable TopoIIα inhibitory as well as antiproliferative activities. In particular, 2PBFs possess divergent mechanism of action on inhibiting DNA topoisomerase IIα from 2PNs, switching Topo poisons 2PNs to Topo catalytic inhibitors 2PBFs. These results suggest that the chromophore scaffold replacement drug design strategy used in this study may result in a change of the binding sites of inhibitors to TopoIIα. This study provides insights to the structure optimization of DNA topoisomerase IIα inhibitors.

About The Author

Dr. Yuemao Shen received his B. Sc. degree in Chemistry from Anhui Normal University in 1986, M.Sc. in natural products chemistry from Kunming Institute of Botany (KIB) of Chinese Academy of Sciences in 1989. He went to Floss Lab at the University of Washington (UW) as a visiting Scientist and became a joint Ph. D. student of KIB and UW in 1995, and received his Ph. D. degree in Botany from KIB of Chinese Academy of Sciences in 1999. Before joining the faculty of Shandong University in 2010, he was an intern Researcher (1989-1991), assistant Researcher (1991-1995), associate Researcher (1998-1999) and Researcher (1999-2004) in KIB, and Professor in Xiamen University (2004-2010). Being a professor of Shandong University, Dr. Shen’s research interests focus on isolation, structure elucidation, and biosynthesis of natural products with anti-infective or antitumor activities.

About The Author

Miss Huilin Hao received her B. Sc. degree in Pharmacy from Shandong University in 2013. She is currently a graduate student under the supervision of Prof. Yuemao Shen at Shandong University School of Pharmaceutical Sciences. Her research interest includes antitumor mechanisms of topoisomerase inhibitors, endocytic vesicle trafficking and cancer cell metabolism.

About The Author

Dr. Wang Chen received his B. Sc. degree in Pharmacy from Xi’an Jiaotong University Health Sciences Center in 2010, and obtained his Ph. D. degree in Medicine from Shandong University School of Pharmaceutical Sciences in 2015. He is currently a lecturer in Shaanxi University of Technology Vitamin D Research Institute. His research interest is the synthesis of Vitamin D derivatives.

Journal Reference

Eur J Med Chem. 2015;102:277-87.

Hao H¹, Chen W¹, Zhu J², Lu C¹, Shen Y³.

Show Affiliations

1. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China.
2. State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China.
3. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China; State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China. Electronic address: yshen@sdu.edu.cn.

Abstract

Eight 2-phenylnaphthalenoids (2PNs) (3a-h) and twenty four 2-phenylbenzofuranoids (2PBFs) (4a–4j, 5a-5j, 6a, 6f-6h) were successfully designed, synthesized and their antiproliferative and in vitro DNA topoisomerase inhibitory activities were evaluated. Nine compounds (four 2PNs and five 2PBFs) showed either TopoI or TopoIIα inhibitory activities. Six compounds (four 2PNs and two 2PBFs) exhibited potent cytotoxicity with IC50 values for 72 h exposure ranging from 0.3 to above 20 μM against MDA-MB-231, MDA-MB-435, HepG2 and PC3 cell lines. The two 2PBFs displayed comparable and even better antiproliferative as well as TopoIIα inhibitory activities than 2PNs. Interestingly, the active 2PBFs displayed different mechanisms of TopoIIα inhibition from that of 2PNs, suggesting that the chromophore scaffold replacement may result in a change of the binding site of inhibitors to TopoIIα. Furthermore, the mechanisms of antiproliferation on MDA-MB-231 cells indicate that compounds 5a and 5f are promising for further development of anticancer agents. The results of this study reveal that the evolutionary strategy of medicinal chemistry through scaffold hopping is a promising strategy for structure optimization of TopoIIα inhibitors.

Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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