The antimicrobial peptide pardaxin exerts potent anti-tumor activity against canine perianal gland adenoma.

Pardaxin is an antimicrobial peptide of 33 amino acids, originally isolated from marine fish. We previously demonstrated that pardaxin has anti-tumor activity against murine fibrosarcoma, both in vitro and in vivo. In this study, we examined the anti-tumor activity, toxicity profile, and maximally-tolerated dose of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resection of canine histiocytomas revealed large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. Pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our findings indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data justify the veterinary application of pardaxin, and also provide invaluable information for veterinary medicine and future human clinical trials.

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells.

Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15µg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.

Synthesis of a shark repellent peptide toxin, Pardaxin (16-33) on a highly flexible polymer support: CLPSER.

A high swelling resin, CLPSER has been developed and utilized for the solid phase synthesis of Pardaxin, which is an 18-residue peptide. The resin was characterized by gel phase (13)C NMR, IR and SEM. The utility of the new polymer support in polypeptide synthesis was further established by the comparative synthesis of pardaxin with commercially available Merrifield resin. The MALDI TOF MS, amino acid analysis and the HPLC revealed the superior quality of CLPSER.

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells.

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the alpha-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran -2- one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1alpha and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.

pH-dependent pore formation properties of pardaxin analogues.

The interaction of pardaxin, a shark-repellent neurotoxin, and its charge-modified analogues with vesicles and human erythrocytes is described. The following six analogues and derivatives were synthesized by a solid phase method: [Glu8, Glu16]pardaxin, [N1-succinamido,Glu8,Glu16]pardaxin, [N1,Lys8,Lys16-triacetyl]pardaxin, des-[1----9]pardaxin (Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202-20209), and des-[1----9] [Glu16]pardaxin. The relative hydrophobic characteristics of the analogues were examined using reverse-phase high performance liquid chromatography. The pH-dependent spectroscopic and functional characteristics of the analogues were also investigated at either neutral or acidic pH. Spectroscopic characterization was achieved by measuring circular dichroism both before and after binding to vesicles, at either neutral or acidic pH. The ability of the peptides to dissipate a diffusion potential, to cause calcein release or the pH-dependent release of 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt/p-xylene-bis[pyridinium bromide] from sonicated unilamellar liposomes, as well as measurements of cytolytic activity on human erythrocytes, served to functionally characterize the peptides. We show a direct correlation between alpha-helical content, the analogues' hydrophobicity, and their pore-forming properties at the different pH values tested. We also demonstrate that the charge of the N terminus and of the peptide backbone, but not of the C terminus, affects the secondary structure as well as the activities of the analogues. Finally, we show that the cytolytic activity of pardaxin at neutral pH is not retained by any of the analogues.

Solution structure of pardaxin P-2.

Pardaxin is a mucosal secretion of the Pacific sole Pardachirus pavoninus that exhibits unusual shark repellent and surfactant properties [Thompson, S. A., Tachibana, K., Nakanishi, K., & Kubota, I. (1986) Science 233, 341-343]. This 33 amino acid polypeptide folds into ordered structures in trifluoroethanol-water solution and in micelles but adopts a random-coiled structure in water solution. The complete proton NMR spectrum of pardaxin P-2 has been assigned in CF3CD2OD/H2O (1:1) solution, and the three-dimensional structure has been elucidated with distance restrained molecular dynamics calculations. It is demonstrated that peptide segments within the 7-11 and 14-26 residue stretches are helical while residues at the C- and N-terminus exist predominantly in extended conformations in solution. The dipeptide 12-13 segment connecting the two helices exists as a bend or a hinge allowing the two helices to be oriented in a L-shaped configuration. These studies establish that pardaxin P-2 adopts a novel amphiphilic helix (7-11)-bend (12-13)-helix (14-26) motif with Pro-13 forming the focal point of the turn or bend between the two helices.

Sequencing and synthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophore activity.

Pardaxin, an amphipathic polypeptide secreted by the Red Sea flatfish Pardachirus marmoratus whose sequence is NH2-G-F-F-A-L-I-P-K-I-I-S-S-P-L-F-K-T-L-L-S-A-V-G-S-A-L-S-S-S-G-G-Q-E, was synthesized by the solid-phase method. The structure was verified by sequencing. The synthetic polypeptide changed the resistance of lipid bilayers by forming pores. At 10(-7)-10(-8) M, the synthetic pardaxin increased the frequency of the spontaneous release of quanta of acetylcholine at the neuromuscular junction by up to 100-fold, resembling the native product. Synthetic pardaxin seems to be a suitable tool for investigating the molecular structures underlying channel selectivity.