The zebrafish embryo as a tool for screening and characterizing pleurocidin host-defense peptides as anti-cancer agents

The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at 6432 &\u3bc\u3c5;g/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 &\u3bc\u3c5;g/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 &\u3bc\u3c5;M; ~64 &\u3bc\u3c5;g/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 &\u3bc\u3c5;M peptide. Four other peptides killed embryos after 24 hours of exposure at 1 &\u3bc\u3c5;M. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses ( 655 &\u3bc\u3c5;M). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.Peer reviewed: YesNRC publication: Ye

Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori, Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 μM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(−)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(−) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the “activated cofactor” of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance

Reciprocal Expression of Integration Host Factor and HU in the Developmental Cycle and Infectivity of Legionella pneumophila▿ †

Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Δihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under Ptac (isopropyl-β-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-β-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Δihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts

Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

<div><p>The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for <i>in vivo</i> toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, <i>in vivo</i> model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an <i>in vivo</i> model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.</p></div

Concentration response profiles of zebrafish larvae exposed to MBV condensate from 6 to 72 hpf.

<p><b>A</b>) DMSO Carrier control (CC); <b>B-H</b>) 10, 15, 20, 25, 30, 40, 50 µg/mL CSC, respectively. Scale bars correspond to 280 µm.</p

Comparison of the toxicity profile of zebrafish larvae following exposure to cigarette smoke condensates (CSCs).

<p>Concentration response (ug/ml) of CSCs prepared using Health Canada Intense (HCI) smoking regime. Asterisk indicates no significant difference between cigarette types for the specific toxic parameter measured (f-test, p>0.05).</p><p>Comparison of the toxicity profile of zebrafish larvae following exposure to cigarette smoke condensates (CSCs).</p

Behavioural toxicity following exposure to the MBV condensate from 72–120 hpf.

<p>A) Larval response to 5 minute cycles of light-dark. B) Dark startle response presented as the difference between the light and dark response levels. C) Concentration response curve for dark response.</p

Cerebal hemorrhaging in zebrafish larvae exposed to BSV condensate from 6–72 hpf.

<p><b>A</b>) DMSO Carrier control (CC); <b>B</b>) 12.5 µg/mL CSC. <b>C</b>) 20 µg/mL CSC. Arrows indicate blood pooling. Scale bars correspond to 280 µm.</p

Changes in heart rate following larval exposure to cigarette smoke condensates (CSCs) from 24–48 hpf.

<p>CSCs were prepared under the International Organization for Standardization (ISO) and Health Canada Intense (HCI) smoking regimes. bpm = beats per minute.</p><p><i>Note</i>. Concentrations of CSC: ^a25 µg/mL; ^b50 µg/mL; ^c30 µg/mL; ^d15 µg/mL; ^e20 µg/mL; ^f40 µg/mL.</p><p>Changes in heart rate following larval exposure to cigarette smoke condensates (CSCs) from 24–48 hpf.</p