The Bi-(AID-1-T) G-Quadruplex Has a Janus Effect on Primary and Recurrent Gliomas: Anti-Proliferation and Pro-Migration.

High-grade gliomas are considered an incurable disease. Despite all the various therapy options available, patient survival remains low, and the tumor usually returns. Tumor resistance to conventional therapy and stimulation of the migratory activity of surviving cells are the main factors that lead to recurrent tumors. When developing new treatment approaches, the effect is most often evaluated on standard and phenotypically depleted cancer cell lines. Moreover, there is much focus on the anti-proliferative effect of such therapies without considering the possible stimulation of migratory activity. In this paper, we studied how glioma cell migration changes after exposure to bi-(AID-1-T), an anti-proliferative aptamer. We investigated the effect of this aptamer on eight human glioma cell cultures (Grades III and IV) that were derived from patients' tumor tissue; the difference between primary and recurrent tumors was taken into account. Despite its strong anti-proliferative activity, bi-(AID-1-T) was shown to induce migration of recurrent tumor cells. This result shows the importance of studying the effect of therapeutic molecules on the invasive properties of glioma tumor cells in order to reduce the likelihood of inducing tumor recurrence.

Reparative properties of human glioblastoma cells after single exposure to a wide range of X-ray doses.

Radiation therapy induces double-stranded DNA breaks in tumor cells, which leads to their death. A fraction of glioblastoma cells repair such breaks and reinitiate tumor growth. It was necessary to identify the relationship between high radiation doses and the proliferative activity of glioblastoma cells, and to evaluate the contribution of DNA repair pathways, homologous recombination (HR), and nonhomologous end joining (NHEJ) to tumor-cell recovery. We demonstrated that the GO1 culture derived from glioblastoma cells from Patient G, who had previously been irradiated, proved to be less sensitive to radiation than the Sus\fP2 glioblastoma culture was from Patient S, who had not been exposed to radiation before. GO1 cell proliferation decreased with radiation dose, and MTT decreased to 35% after a single exposure to 125 Gγ. The proliferative potential of glioblastoma culture Sus\fP2 decreased to 35% after exposure to 5 Gγ. At low radiation doses, cell proliferation and the expression of RAD51 were decreased; at high doses, cell proliferation was correlated with Ku70 protein expression. Therefore, HR and NHEJ are involved in DNA break repair after exposure to different radiation doses. Low doses induce HR, while higher doses induce the faster but less accurate NHEJ pathway of double-stranded DNA break repair.

A Combined Effect of G-Quadruplex and Neuro-Inducers as an Alternative Approach to Human Glioblastoma Therapy.

Cancer cell reprogramming based on treatment with G-quadruplex, having antiproliferative power, along with small molecules able to develop iPSCs into neurons, could create a novel approach to diminish the chance of glioblastoma recurrence and circumvent tumor resistance to conventional therapy. In this research, we have tested several combinations of factors to affect both total cell cultures, derived from tumor tissue of patients after surgical resection and two subfractions of this cell culture after dividing them into CD133-enriched and CD133-depleted populations (assuming CD133 to be a marker of glioblastoma stem-like cells). CD133+ and CD133- cells exhibit different responses to the same combinations of factors; CD133+ cells have stem-like properties and are more resistant. Therefore, the ability to affect CD133+ cells provides a possibility to circumvent resistance to conventional therapy and to build a promising strategy for translation to improve the treatment of patients with glioblastoma.

Neuroinductive properties of mGDNF depend on the producer, E. Coli or human cells.

The glial cell line-derived neurotrophic factor (GDNF) is involved in the survival of dopaminergic neurons. Besides, GDNF can also induce axonal growth and creation of new functional synapses. GDNF potential is promising for translation to treat diseases associated with neuronal death: neurodegenerative disorders, ischemic stroke, and cerebral or spinal cord damages. Unproductive clinical trials of GDNF for Parkinson's disease treatment have induced to study this failure. A reason could be due to irrelevant producer cells that cannot perform the required post-translational modifications. The biological activity of recombinant mGDNF produced by E. coli have been compared with mGDNF produced by human cells HEK293. mGDNF variants were tested with PC12 cells, rat embryonic spinal ganglion cells, and SH-SY5Y human neuroblastoma cells in vitro as well as with a mouse model of the Parkinson's disease in vivo. Both in vitro and in vivo the best neuro-inductive ability belongs to mGDNF produced by HEK293 cells. Keywords: GDNF, neural differentiation, bacterial and mammalian expression systems, cell cultures, model of Parkinson's disease.

RNA Aptamers for Theranostics of Glioblastoma of Human Brain.

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2'-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer - EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

Protective effects of Lactobacillus fermentum U-21 against paraquat-induced oxidative stress in Caenorhabditis elegans and mouse models.

The aims of this work were to identify in vivo manifestations of antioxidant activity of Lactobacillus strains isolated from healthy human biotopes and to show the possibility of protective action of the selected strain on the model of oxidative stress induced by paraquat in the model of early Parkinson's disease (PD) in mice. We studied the protective effects of 14 Lactobacillus strains belonging to five species on the lifespan of the soil nematode Caenorhabditis elegans experiencing oxidative stress induced by paraquat. The Lactobacillus strains used in this study were selected previously based on their ability to reduce oxidative stress in vitro. One of the strains that showed promising results on C. elegans was tested in a mouse model of PD in which C57/BL6 mice were injected regularly with paraquat. We assessed the state of their internal organs, the preservation of dopaminergic neurons in the substantia nigra as well as their motor coordination. The positive impact of Lactobacillus fermentum U-21 strain supplementation on paraquat treated animals was observed. L. fermentum U-21 strain reduced the toxicity of paraquat in C. elegans model: the lifespan of the soil nematode C. elegans was extended by 25%. L. fermentum U-21 protected the mice against anatomical and behavioral changes typical of PD: there were no changes in the coordination of movement and the preservation of dopaminergic neurons in the brain. Life span of the nematode C. elegans pre-grown on a lawn of E. coli OP50 + Lactobacillus under oxidative stress conditions; the concentration of the oxidizing agent paraquat in the S medium was 50 mmol l-1.

Bimodular Antiparallel G-Quadruplex Nanoconstruct with Antiproliferative Activity.

Oligonucleotides with an antiproliferative activity for human cancer cells have attracted attention over the past decades; many of them have a G-quadruplex structure (GQ), and a cryptic target. In particular, DNA oligonucleotide HD1, a minimal GQ, could inhibit proliferation of some cancer cell lines. The HD1 is a 15-nucleotide DNA oligonucleotide that folds into a minimal chair-like monomolecular antiparallel GQ structure. In this study, for eight human cancer cell lines, we have analyzed the antiproliferative activities of minimal bimodular DNA oligonucleotide, biHD1, which has two HD1 modules covalently linked via single T-nucleotide residue. Oligonucleotide biHD1 exhibits a dose-dependent antiproliferative activity for lung cancer cell line RL-67 and cell line of central nervous system cancer U87 by MTT-test and Ki-67 immunoassay. The study of derivatives of biHD1 for the RL-67 and U87 cell lines revealed a structure-activity correlation of GQ folding and antiproliferative activity. Therefore, a covalent joining of two putative GQ modules within biHD1 molecule provides the antiproliferative activity of initial HD1, opening a possibility to design further GQ multimodular nanoconstructs with antiproliferative activity-either as themselves or as carriers.

Time course of transient expression of pre-α-pro-GDNF and pre-ß-pro-GDNF transcripts, and mGDNF mRNA region in Krushinsky-Molodkina rat brain after audiogenic seizures.

The expression of glial cell line-derived neurothrophic factor (GDNF) transcript forms pre-(α)pro-gdnf, pre-(ß)pro-gdnf, and their common region m-gdnf in the pons as well as the inferior (IC) and superior colliculi in Krushinsky-Molodkina (KM) rats and in the strain "0" was analyzed by quantitative real-time polymerase chain reaction (PCR) in the control (unstimulated KM and "0" rats) and 1.5, 4.5, and 8 h after auditory stimulation. Such stimulation induced audiogenic seizures (AS) in KM rats. Audiogenic seizure was not observed in "0" rats, which was obtained by selection for the absence of AS in a population of F2 hybrids between KM and Wistar rats not predisposed to AS. A significant drop in the level of all transcripts was observed 1.5 h after auditory stimulation in both KM and "0" rats. In most cases, the average expression of α and ß isoforms and m-region 4.5 h after stimulation was greater than those after 1.5 and 8 h. At the same time, the expression of pre-(ß)pro-gdnf in the IC of KM rats 4.5 h after the stimulation was significantly lower than after 1.5 or 8 h. This work presents the first demonstration of different time courses of expression of the α and ß GDNF isoforms during physiological processes in genotype-specific pathology.

The Evaluation of Pharmacodynamics and Pharmacokinetics of Anti-thrombin DNA Aptamer RA-36.

Anticoagulants are a vital class of drugs, which are applied for short-term surgical procedures, and for long-term treatments for thrombosis prevention in high risk groups. Several anticoagulant drugs are commercially available, but all have intrinsic disadvantages, e.g., bleeding risks, as well as specific ones, e.g., immune response to peptide/protein drugs. Therefore, the search for novel, efficient and safe anticoagulants is essential. Nucleic acid aptamers are an emerging class of contemporary pharmaceuticals which are fully biocompatible and biodegradable; they have low toxicity, and are as efficient as many protein-based drugs. The anti-thrombin DNA aptamer RA-36 has been created using a combination of rational design and molecular dynamics, showing several extra-features over existing aptamers. Aptamer RA-36 has a bimodular structure; the first G-quadruplex binds and inhibits thrombin, whereas the second G-quadruplex varies the properties of the first. This bimodular structure provides a favorable dose-effect dependence allowing the risk of bleeding to be potentially decreased. Here, the results of efficiency trials of the aptamer are presented. The aptamer RA-36 has a distinctive species specificity; therefore, the careful selection of experimental animals was required. The anticoagulant activity was characterized in rats and monkeys in vivo. Antithrombotic activity was evaluated in the live murine model of the induced thrombosis. Pharmacokinetics was estimated by tracking radionuclide labeled aptamer in rats. The aptamer was thoroughly characterized using bivalirudin as a reference drug. Despite the different profiles of anticoagulant activity, these two compounds could refer to each other, and the corresponding doses could be estimated. Bivalirudin turned out to have 10-fold higher anticoagulant and antithrombotic activity. The difference in activity is easy to explain due to the pharmacokinetic profiles of the substances: the aptamer RA-36 has 20-fold faster elimination from blood with a half-life of 1 min. The entire dataset revealed that the non-modified DNA aptamer could be an alternative to the currently used bivalent peptide inhibitor; the dosage profile could be improved by manipulating aptamer pharmacokinetics. The study has revealed aptamer RA-36 to be one of the most promising candidates for further development as a new generation of anticoagulants.

Hiding in the Shadows: CPOX Expression and 5-ALA Induced Fluorescence in Human Glioma Cells.

Protoporphyrin IX (PpIX) is widely used in photodynamic diagnosis. To date, the details of molecular mechanisms underlying PpIX accumulation in malignant cells after 5-ALA administration remain unclear. The fluorescence of PpIX was studied in human glioma cells. Several cell cultures were established from glioma tumor tissue to study the differences between fluorescence-positive and fluorescence-negative human glioma tumors. The cell cultures demonstrated fluorescence profiles similar to those of source tumor tissues, which allows us to use these cultures in experimental research. Dynamics of the rates of synthesis and degradation of fluorescent protoporphyrin IX was studied in the cultures obtained. In addition, the expression of CPOX, an enzyme involved in PpIX synthesis, was evaluated. mRNA levels of heme biosynthesis enzymes were analyzed, and PpIX fluorescence proved to correlate with the CPOX protein level, whereas no such correlation was observed at the mRNA level. Fluorescence intensity decreased at low levels of the enzyme, which indicates its critical role in PpIX fluorescence. Finally, the fluorescence intensity proved to correlate with the proliferative activity.

Availability of Pre- and Pro-regions of Transgenic GDNF Affects the Ability to Induce Axonal Sprout Growth.

Plasmids containing four GFP-tagged isoforms of the human GDNF gene, with both pre- and pro-regions (pre-pro- GDNF), with the pre- (pre-GDNF) or the pro-region (pro-GDNF) alone, and without the pre- and pro-regions (mGDNF), were used to transfect HEK293 cells (human embryonic kidney cell line). The effect of the transgenic products on the growth of processes was studied in the spinal ganglia of 14-day rat embryos. Media conditioned by the transgenic cells were used to culture explants and dissociated cells of embryonic dorsal root ganglia attached to the bottom of the plate. Medium conditioned by gfp-transgenic HEK293 cells was used as the control. Spinal ganglia explants and dissociated cells cultured in a medium supplemented with recombinant GDNF (recGDNF) as well as in conditioned media containing the pre-GDNF and mGDNF products demonstrated active growth of processes immunopositive for neuronal marker beta-3-tubulin as early as on culture day 4. The ganglia and cells cultured in control medium and media conditioned by cells transgenic for pro-GDNF had no or very few processes even after 10 days of culture.

Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

The neuronal insulin sensitizer dicholine succinate reduces stress-induced depressive traits and memory deficit: possible role of insulin-like growth factor 2.

BACKGROUND: A number of epidemiological studies have established a link between insulin resistance and the prevalence of depression. The occurrence of depression was found to precede the onset of diabetes and was hypothesized to be associated with inherited inter-related insufficiency of the peripheral and central insulin receptors. Recently, dicholine succinate, a sensitizer of the neuronal insulin receptor, was shown to stimulate insulin-dependent H2O2 production of the mitochondrial respiratory chain leading to an enhancement of insulin receptor autophosphorylation in neurons. As such, this mechanism can be a novel target for the elevation of insulin signaling. RESULTS: Administration of DS (25 mg/kg/day, intraperitoneal) in CD1 mice for 7 days prior to the onset of stress procedure, diminished manifestations of anhedonia defined in a sucrose test and behavioral despair in the forced swim test. Treatment with dicholine succinate reduced the anxiety scores of stressed mice in the dark/light box paradigm, precluded stress-induced decreases of long-term contextual memory in the step-down avoidance test and hippocampal gene expression of IGF2. CONCLUSIONS: Our data suggest that dicholine succinate has an antidepressant-like effect, which might be mediated via the up-regulation of hippocampal expression of IGF2, and implicate the neuronal insulin receptor in the pathogenesis of stress-induced depressive syndrome.

Distant cortical locations of the upper and lower quadrants of the visual field represented by neurons with elongated and radially oriented receptive fields.

In our previous study of the cytoarchitectonic field 7 of cat cortex we had described neurons with extremely elongated receptive fields (RFs). The long axes of these RFs were oriented radially, towards the centre of the retina. These neurons represented only the lower contralateral part of visual field. They were surrounded from all sides by neurons with clearly different RF properties. We proposed that neurons with a similar radial organization and with RFs in the upper visual field also exist in the cortex but are localized in the area that was distant from the representation of the corresponding lower visual field. We expected to find these neurons in front of the representation of the upper visual field in areas V1, V2 and V3 (fields 17, 18 and 19), behind the central representation in area 21a. This cortical region was studied in five behaving cats. In all animals, neurons with radial RFs in the upper visual field were found in the expected location. As in the lower visual field, their RFs always spared the central visual field. Other RF properties of these neurons were also very similar to those found previously in the lower visual field. It became obvious that neurons with radial RFs are included into the fourth extrastriate crescent with complete contralateral representation. However, in the fourth crescent, RF properties in the central visual field differed significantly from those on the periphery. As a result, neurons with similar radial RFs in the upper and lower visual fields were located in the distant cortical regions, and were separated by the representation of the central visual field presented by the non-radial neurons of the cytoarchitectonic area 21a.

The influence of donor age, nerve growth factor, and cografting with drosophila cells on survival of peripherally grafted embryonic or fetal rat dorsal root ganglia.

Previous studies have shown that embryonic rat and human dorsal root ganglion (DRG) cells survive grafting to the cavity of extirpated adult rat DRG. Furthermore, grafted human embryonic neurons were shown to send axons peripherally and into the spinal cord, where they establish functional synaptic connections. This study analyzed the survival of orthotopically allografted rat DRG cells from embryonic stages 15 (E15) and 20 (E20), and the influence on their survival of nerve growth factor (NGF). NGF was delivered to the DRG transplants either by pump infusion or by cotransplantation of cells from Drosophila melanogaster, transgenic for human NGF. Lumbar DRGs of adult rats were removed and a collection of E15 or E20 DRGs placed in the cavity. One month after grafting the total number of DRG cells in the grafts was counted. Differentiation of subpopulations of DRG cells was estimated by counting cells immunostained for calcitonin gene-related peptide (CGRP), Griffonia simplicifolia agglutinin isolectin B4 (GSA), or heavy neurofilament protein (antibody RT97). The results show: i) similar survival of E15 and E20 grafts, with great variability in the survival of different subpopulations in E15 transplants, but a more consistent distribution of different phenotypes in E20 transplants; ii) infusion of NGF for 2 weeks increases the survival of E15 transplants, but has a negative effect on E20 transplants; iii) Drosophila cells transfected with human NGF gene survive peripheral xenografting and have a positive effect on the survival of the GSA- and CGRP-positive populations in E15 and E20 transplants; iv) Drosophila cells without the human NGF gene increase cell survival in E20 transplants. These data suggest that i) the effect of NGF is dependent on the embryonic stage of the transplants, ii) age-dependent sensitivity to NGF influences graft survival, and iii) transgenic Drosophila cells can be cotransplanted with embryonic neural tissue to the mammalian peripheral nervous system with a positive effect on the survival of neural grafts.