Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material.

Interest in cell-penetrating peptides (CPPs) as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue) for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin--galactosidase (ABG), which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases.

Design, synthesis and properties of novel powerful antioxidants, glutathione analogues.

Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC(50) of GSH: 1,231.0 +/- 311.8 microM; EC(50) of UPF peptides: from 0.03 to 35 microM) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC(50): 11.4 +/- 1.0 microM). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 microM concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.

Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide.

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide-MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 microM, the peptide-MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50's were determined for MTX, MTX-YTA2 and YTA2 to be 18.5, 3.8 and 20 microM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

Cell-penetrating peptides: a comparative membrane toxicity study.

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43-58), pTAT(48-60), pVEC(615-632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43-58), pTAT(48-60), and pVEC(615-632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 microM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.

Protein cargo delivery properties of cell-penetrating peptides. A comparative study.

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.

Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA.

In this work, we have targeted positions 18-38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24-38 (EC50=70 nM) or 27-38 (EC50=80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Amyloid precursor protein carboxy-terminal fragments modulate G-proteins and adenylate cyclase activity in Alzheimer's disease brain.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer's disease (AD) and age-matched control brains was compared. 10 microM APP(639-648)-APP(657-676) (PEP1) causes a fivefold stimulation in the [35S]GTPgammaS-binding to control hippocampal G-proteins. APP(657-676) (PEP2) and APP(639-648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649-669) (PEP3) showed no regulatory activity in the [35S]GTPgammaS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [35S]GTPgammaS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

Anticonvulsant activity of a nonpeptide galanin receptor agonist.

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace (125)I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace (125)I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.