Prostones as ClC-2 Channel Activators for Treatment of Diseases and Disorders. CUPPOLETTI J1 and UENO R2 1University of Cincinnati College of Medicine, Cincinnati, OH USA; and 2Sucampo Pharmaceuticals Inc, Bethesda, MD USA
Background: Prostones are a class of bicyclic fatty acids. One of these, lubiprostone is approved for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C) in the United States.
Methods & Results: Lubiprostone activates Cl- currents, thus increasing salt and water flow in the small intestine. Electrophysiological studies of epithelial tissues show that Cl- currents are increased by low concentrations (<100 nM) lubiprostone. These Cl- currents persist with knockdown of the cystic fibrosis transmembrane regulator (CFTR), another epithelial Cl- channel. However, the lubiprostone-activated Cl- currents are ablated by knockdown of ClC-2. Recombinant human ClC-2 is activated in a dose-dependent manner by lubiprostone (EC50 = 20 nM). Recombinant human CFTR was not activated at concentrations as high as 1 µM. Lubiprostone increased single 3-4 pS Cl- channel activity of both human and Xenopus ClC-2 in the apical membrane of the cells, at concentrations <100 nM. This is consistent with apical membrane localization of ClC-2 in T84 and Caco-2 human intestinal cell lines using nystatin permeabilization approaches. This is also consistent with animal studies showing increased salt and water transport into the intestinal lumen with lubiprostone. ClC-2 activation by lubiprostone does not involve increases in intracellular cAMP or Ca2+, activation of prostaglandin receptors or phosphorylation by PKA. In Caco-2 cells and porcine ileum ClC-2 is present at the tight junctions. In porcine ileum, lubiprostone appears to promote repair of epithelial barrier function impaired by ischemia, accompanied by movement of ClC-2 to the tight junctions, a mechanism which may contribute to the treatment of IBS-C.
Conclusions: 1. Various studies have demonstrated that lubiprostone is a Cl- channel activator that activates ClC-2 in the apical membrane of intestinal cells. 2. ClC-2 activation may be beneficial in diseases and conditions where tight junction integrity is compromised. 3. ClC-2 activation by prostones is a useful therapeutic tool in GI disorders. Supported by a grant from Sucampo Pharmaceuticals Inc.
Authors’ disclosure statement: Both authors have significant financial support from Sucampo Pharmaceuticals Inc.
according to registration: Curkovic Perica Auxins: Effects on Bacteria and Tumours PERICA MC1, ESTER K2, KRALJ M2 1Univ. of Zagreb, Faculty of Science, 2Rudjer Boskovic Institute, Bijenicka 54, Zagreb, Croatia
Background: Phytohormones auxines are a group of molecules involved in mediating a number of essential plant growth and developmental processes. These plant hormones are not only synthesized by higher plants, but also by lichens, mosses, fungi and bacteria. Furthermore, not only bacterial species that live in soil and/or interact with plants produce phytohormons. Some human-associated and pathogenic bacteria produce auxins, too. It was shown that indoleacetic acid (IAA), as the most widespread natural auxin present in most living organisms, can trigger alterations in the main metabolic pathways of bacterial, yeast and human cells.
Methods:Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were used to treat phytoplasmas and tumours. Phytoplasma-infected plants were maintained on Murashige and Skoog medium supplemented with IAA (11 or 57μM) or IBA (2.5, 4.9, 9.8 or 19.7 μM). Randomly chosen shoots from each treatment were tested for the presence of phytoplasmas by nested polymerase chain reaction amplifying highly conserved phytoplasmal 16S rRNA gene. Cytotstatic activity on tumour cells was assessed on 5 human cell lines, which are derived from 4 cancer types: MCF-7 (breast carcinoma), SW 620 (colon carcinoma), HCT 116 (colon carcinoma), MOLT-4 (acute lymphoblastic leukemia), H 460 (lung carcinoma). IAA and IBA were added to the cell culture medium in five, 10-fold dilutions (10-7 to 10-3 M) and incubated for 72 hours. The cell growth rate was evaluated by performing the MTT assay, which detects dehydrogenase activity in viable cells.
Results: The mechanism of auxins effect on phytoplasmas (mycoplasma-like organisms, economically important plant pathogenic bacteria), involves changes in plant-host metabolism and gene expression. It was shown that phytoplasma-free plants can be obtained upon treatment of infected plants with IBA, while treatment with IAA induced recovery of plants, but the pathogen was still present in the plant tissue. IAA also inhibited the growth of tumor cells (IC50 ≈ 500 μM), whereby the breast cancer cell line MCF-7 was the most sensitive cell line. Thus both auxines could be further evaluated as novel anticancer therapeutics.
Conclusions: Auxins exhibit both antibacterial and antitumour activity.
Spontaneous Formation of L-isoAspartate and Gain-of-Function in Fibronectin CURNIS F, LONGHI R1, CRIPPA L, CATTANEO A, DONDOSSOLA E, BACHI A, CORTI A
DIBIT-Department of Oncology, CIGT Program and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, Milan, Italy; 1Istituto di Chimica del Riconoscimento Molecolare, CNR, Milan, Italy.
Isoaspartate formation in extracellular matrix (ECM) proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically “repaired” to RGD by protein-L-isoAsp-O-methyltransferase (PIMT). We show here that isoaspartate formation is also a mechanism for ECM activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an v3-integrin binding site containing the L-isoDGR sequence, that is enzymatically “deactivated” to DGR by PIMT. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates v3 antagonists (named “isonectins”) that competitively bind RGD-binding sites and inhibit endothelial cell adhesion, proliferation and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular-clock for activating latent integrin binding sites in proteins.
1DIBIT-Department of Oncology, CIGT Program and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, Milan, Italy; 2Istituto di Chimica del Riconoscimento Molecolare, CNR, Milan, Italy; 3MolMed SpA, Milan, Italy.
Various peptides containing the NGR motif have been discovered by selecting peptide-phage display libraries in tumor-bearing animals. These peptides can home to tumor neovasculature by binding aminopeptidase N (CD13), a marker of angiogenic vessels. We have previously demonstrated that NGR peptides can be exploited for ligand-directed delivery of cytokines, e.g. TNF and interferon-, to CD13-positive tumor blood vessels. One of these conjugates, made of CNGRC fused to tumor necrosis factor-a (NGR-TNF), is now under investigation in phase II clinical studies for cancer treatment, highlighting the value of CD13-targeting peptides in drug development. We have also found that NGR can rapidly convert to isoDGR and DGR by asparagine deamidation. In vitro and in vivo studies have shown that isoDGR, but not DGR, can bind v3 integrin in the tumor neovasculature. Pharmacological studies performed in tumor-bearing animal models showed that low doses (picograms) of NGR-TNF or isoDGR-TNF fusion proteins are sufficient to induce anti-tumor effects, by virtue of a targeting mechanism, when administered alone or in combination with chemotherapy. Considering that CD13 and v3 integrin are markers of angiogenic blood vessels, natural or synthetic polypeptides containing the NGR or the isoDGR motif may be exploited as ligands for targeted delivery of cytokines, nanoparticles, genes or imaging compounds to angiogenic vasculature in tumors.
