Haloperidol,but not olanzapine,may affect expression of PER1 and CRY1 genes in human glioblastoma cell line

Background: There is barely any evidence of antipsychotic drugs affecting the molecular clockwork in human, yet it is suggested that clock genes are associated with dopaminergic transmission, i.e. the main target of this therapeutics. We decided to verify if haloperidol and olanzapine affect expression of CLOCK, BMAL1, PER1 and CRY1 in a human central nervous system cell line model. Methods: U-87MG human glioblastoma cell line was used as an experimental model. The cells were incubated with or without haloperidol and olanzapine in the concentration of 5 and 20 μM for 24 h. Real-time quantitative polymerase chain reaction with the ΔC_T analysis was used to examine the effect of haloperidol and olanzapine on the mRNA expression of the genes. Results: At 5 μM, haloperidol decreased expression of CRY1 almost 20-fold. There was nearly a 1.5-fold increase in expression of PER1. Considering the 20 μM haloperidol concentration and both olanzapine concentrations, no other statistically significant effect was observed. Conclusions: At certain concentration, haloperidol seems to affect expression of particular clock genes in a human central nervous system cell line model, yet mechanism underlying this phenomenon remains elusive.     

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.     

The influence of nitric oxide on the regulation of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator expression

Nitric oxide produced in various human tissues by nitric oxide synthase is involved in the regulation of many physiological processes. Mechanism of its action is diverse. The most important physiological activity of nitric oxide is guanylate cyclase activation and an increase of cGMP synthesis. At low concentrations NO plays a pivotal role in vessel relaxation and possesses antithrombotic, antiproliferative and anti-inflammatory features as well. An excessive production of nitric oxide can disturb vascular hemostasis and contribute to development of cardiovascular diseases. Studies provide that NO also participate in fibrynolysis regulation by the influence on the PAI-1 and t-PA expression, what may have important clinical implications. The aim of this review is to present current knowledge about the role of nitric oxide in the regulation of these plasminogen activation system factors.     

Significance of MDM2 protein in the cell cycle

The basis of oncogenesis underlies the modification of the control of the cell cycle, which leads to disturb balance between proliferation and apoptosis. The MDM2 protein suppresses the ability of p53 to activate genes responsible for repairing or apoptosis, but also promotes p53 degradation by ubiquitination. MDM2 inhibits tumor suppressor property of pRb, by releasing E2F1, which stimulates DNA synthesis in S-phase. MDM2 influences on the neuronal and muscle differentiation. Quantity and stability of the MDM2 protein is regulated by p73, p53, TSG101, p14ARF and Ras-Raf-MEK-ERK pathway. Changes of the level of the MDM2 can disturb control of cell cycle and contribute to oncogenesis.     

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.     

Staphylokinase--a specific plasminogen activator

Staphylokinase is a 135 amino acid protein produced by certain strains of Staphylococcus aureus. It belongs to fibrin-specific plasminogen activator. Staphylokinase converts plasminogen--the inactive proenzyme--to the plasmin, which dissolves the fibrin of a blood clots. This review will focus on the biochemical and thrombolytic properties of staphylokinase and its derivatives, which would make use of treatment in acute myocardial infarction and other cardiovascular diseases.     

The role of functional single nucleotide polymorphisms of the human glucocorticoid receptor gene NR3C1 in Polish patients with bronchial asthma

N363S and ER22/23EK polymorphisms observed within glucocorticoid receptor gene (NR3C1) may play an important role in the development of bronchial asthma. NR3C1 gene is associated with an altered sensitivity to GCs. The aim of the research project was to study the correlation between this NR3C1 gene polymorphisms and occurrence of asthma in the population of Polish asthmatics. Peripheral blood was obtained from 207 healthy volunteers and 221 asthma patients. Genotyping was carried out with PCR-RFLP method. In the groups of patients with uncontrolled moderate asthma and uncontrolled severe disease, the genotype distribution for the investigated polymorphisms was as follows: N363S-AA, AG, GG occurring with 0.881/0.073/0.046 frequency and ER22/23EK-GG, GA, AA occurring with 0.963/0.037/0.000 frequency. Chi-square analysis revealed a significantly different (P < 0.05) distribution between cases and controls for the N363S polymorphisms. The N363S polymorphism of NR3C1 gene is significantly associated with bronchial asthma, susceptibility to the development of moderate to severe form of uncontrolled bronchial asthma.     

The influence of opioid peptides on matrix metalloproteinase-9 and urokinase plasminogen activator expression in three cancer cell lines

Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) regulate proteolysis of the extracellular matrix (ECM) and as a consequence are involved in a number of physiological and pathological states, including cancer. A crucial feature of cancer progression and metastasis is the disruption of the ECM and spreading of proliferating cancer cells. Over-expression of MMPs and uPA is common for most types of cancers and correlates well with the adverse prognosis. Compounds able to modulate the activity of these proteolytic enzymes may become important agents in cancer therapy. In the present study, we examined the effect of the ??-opioid receptor selective peptide, morphiceptin, and its two synthetic analogs on mRNA and protein levels of MMP-9 and uPA in three human cancer cell lines: MCF-7, HT-29, and SHSY5Y. Our findings indicate that in all three cell lines morphiceptin and its analogs attenuated MMP-9 expression and secretion and that this effect is not mediated by opioid receptors but is under control of the nitric oxide system. On the other hand, tested opioids up-regulated uPA levels through a mechanism that involved opioid-receptors. Different pathways by which opioid peptides exert their action in cancer cells can explain their contradictory influence on the level of cancer markers.     

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.