Mariola Piestrzeniewicz

Antidepressant-Like Effect of Endomorphin-1 and Endomorphin-2 in Mice

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are two recently isolated μ-opioid selective peptides with a potent antinociceptive activity, involved in a number of physiological processes, including food intake, vasomotricity, sexual behavior, as well as neuroendocrine and cardiorespiratory functions. The neuroanatomical distribution of endomorphins prompted us to study their antidepressant activity in two animal behavioral models of depression: forced-swimming and tail-suspension tests. In both tests, the intracerebroventricular (i.c.v.) injection of either endomorphin-1 or endomorphin-2 significantly decreased the duration of immobility, interpreted as an expression of ‘behavioral despair’, which could be related to the depression syndrome. These effects of endomorphins did not result from the stimulation of the animal motor activity. We have also demonstrated that the antidepressant-like effect of endomorphins was antagonized by the universal opioid antagonist, naloxone and the μ-opioid receptor selective antagonist, β-funaltrexamine. In contrast, this effect was not antagonized by δ- and κ-opioid receptor selective antagonists, naltrindole and nor-binaltorphimine, respectively. The results of the present study demonstrate that endomorphin-1 and endomorphin-2 produce potent antidepressant-like effects after i.c.v. injection in mice. We may suggest that endomorphins and the μ-opioid receptors might be involved in the physiopathology of depressive disorders, and that the endomorphinergic system could serve as a novel target for the development of antidepressant drugs.

The Influence of Opioids on Urokinase Plasminogen Activator on Protein and mRNA Level in MCF‐7 Breast Cancer Cell Line

Urokinase plasminogen activator plays a key role in tumor‐associated processes, increasing cancer cell invasion and metastasis, and is therefore used as a marker in cancer prognosis. In this study, we have determined the effect of μ‐opioid receptor agonists and antagonists on the urokinase plasminogen activator secretion in MCF‐7 cell line. It was shown that μ‐opioid receptor agonists, such as morphine and endomorphins, greatly stimulate urokinase plasminogen activator secretion, while naloxone and MOR‐selective antagonists elicit the opposite effect. The same tendency was observed also on the urokinase plasminogen activator mRNA level. However, neither agonists nor antagonists had any effect on proliferation of MCF‐7 cells. The findings reported in this study may be useful in designing further experiments aimed at elucidating the role of the opioid system in cancer cells.

Functional Characterization of Opioid Receptor Ligands by Aequorin Luminescence-Based Calcium Assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the μ-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the δ-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the μ- and δ-opioid receptor agonists in both functional assays. The highest potency at the μ-receptor was observed for endomorphin-1, endomorphin-2, and [d-1-Nal3]morphiceptin, whereas deltorphin-II was the most potent δ-receptor agonist. In the aequorin assay, the μ- and δ-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new μ- and δ-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.

Effects of anticancer drugs on transcription in vitro.

Abstract The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E.coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E .coli RNA polymerase (“abortive initiation“); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter ϕ 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G · C ) specificity (actinomycin D), the non-intercalating, d(A · T ) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

Interactions of Novel Morpholine and Hexamethylene Derivatives of Anthracycline Antibiotics with DNA

Abstract Doxorubicin (DOX), daunorubicin (DRB), epidoxorubicin (EDOX) and their analogues with a 3′-NH2 group in daunosamine form a covalent bond with a 2-NH2 group of guanine via a methylene group from formaldehyde (CH2O). It is assumed that a Schiff base type intermediate is formed between CH2O and the 3′-NH2 group in the reaction. This reaction is supposed to occur in the cell. New analogues of anthracyclines with formamidine functionality bound to C-3′ of daunosamine and containing the bulky morpholine (DRBM, DOXM and EDOXM) or hexamethyleneimine rings attached are studied in our laboratory. These substituents decrease the association of the drugs to DNA and potentially hinder the formation of Schiff base-intermediates. Our experiments indicate that the formation of the covalent complexes by DRB, DOX and EDOX under these conditions is confirmed by a high enhancement (17-40x) of the inhibition of overall RNA synthesis by E. coli RNA polymerase on T7 DNA. DRBM and DOXM exhibit a lower enhancement of the inhibition by CH2O (7-13x). The other analogues show a 1.6-3x increase of inhibition. Hence, their covalent binding is lower than that of the parent compounds. These conclusions are confirmed by spectrophotometric estimations following removal of non-covalently associated drugs. Electrophoretic analysis of drug-DNA complexes formed in the presence of CH2O indicates that DRBM and DOXM as their parent compounds induce labile cross-links in DNA. Comparison of the results obtained at the subcellular level with cytotoxicity estimations indicates that there is a correlation between cytotoxicity of the anthracyclines on L1210 cells and transcriptional template activity of drug-DNA complexes formed in the presence of CH2O (r = 0.64; n = 9). These data confirm a notion that covalent attachment of anthracyclines to DNA is an essential event leading to cytotoxicity.

Inhibition of RNA Synthesis in vitro by Acridines - Relation between Structure and Activity

Abstract The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.