Remission of Invasive,Cancer Stem-Like Glioblastoma Xenografts Using Lentiviral Vector-Mediated Suicide Gene Therapy

Background

Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model.

Methodology/Principal Findings

We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect.

Conclusions/Significance

In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.     

Volatile constituents of Erodium cicutarium (L.) L’ Hérit. (Geraniaceae)

Essential oils from Erodium cicutarium were obtained by hydrodistillation (samples consisting of entire plants (ec1), leaves and stems (ec2)) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS), resulting in a total of 177 components being identified. The essential oils were of a very similar chemical composition and consisted mainly of aliphatic compounds and their derivatives. Fatty acids and fatty acid derived compounds were the most common, 51.3% (ec1) and 60.1% (ec2), followed by carotenoid derived compounds, 12.6% (ec1) and 20.2% (ec2), and then terpenoids, 14.9% (ec1) and 14.2% (ec2). The main constituents in the oils were hexadecanoic acid, 22.8% (ec2) and 35.9% (ec1) and hexahydrofarnesyl acetone, 10.8% (ec2) and 11.6% (ec1). The results obtained differ markedly from those previously reported for the same species.     

Comparative study of antioxidant capacity in organs of different Allium species

Cultivated and wild Allium species were investigated in order to compare the antioxidant capacity of their leaves and bulbs. Leaf and bulb anti-oxidative enzymes and scavenger activities, along with quantities of non-enzymic antioxidants, malonyl-dialdehyde and OH radicals were determined. Results obtained suggest that leaves possess higher anti-oxidant and scavenging activities than bulbs in the majority of cultivated and wild Allium species examined. Cultivation of some wild species such as A. flavum L., A. schoenoprasum L. and A. ursinum L. could be conducted in the future in order to produce Allium species with high antioxidant capacity in leaves and bulbs.     

The PECAM-1 gene polymorphism — a genetic marker of myocardial infarction

We investigated a possible association between the C373G (Leu125Val) polymorphism in the platelet endothelial cell adhesion molecule-1 (PECAM-1) and myocardial infarction (MI) among patients with type 2 diabetes (T2DM) in the Slovene population (Caucasians). The study population of this cross-sectional analysis consisted of 452 subjects with T2DM lasting more than 10 years: 142 patients with MI (MI group) and 310 patients (control group) with no history of coronary diseases. There were significant differences of PECAM-1 genotype distribution in patients with MI (CC=28.2%, CG=47.2% and GG=24.6%) compared with subjects in the control group (CC=17.1%, CG=53.5% and GG=29.4%). The multivariate model showed that the CC genotype of the PECAM-1 gene polymorphism (C373G) (OR=1.9, 95% CI 1.2–3.0, P=0.007) was an independent risk factor for MI. The C allele frequency was also significantly higher (P=0.005) in MI (51.8%) than in control subjects (41%). In addition, our study revealed the connection between smoking habits, the duration of diabetes and the total and LDL cholesterol serum levels and MI in Slovene T2DM patients. We suggest that the tested polymorphism of PECAM-1 (C373G) is associated with MI. Therefore, it might be used as genetic marker of MI in T2DM.     

2-(3-Amino-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide: a new tiazofurin analogue with potent antitumour activity

A new tiazofurin analogue, 2-(3-amino-3-deoxy-beta-d-xylofuranosyl)thiazole-4-carboxamide (3), was synthesized starting from d-glucose and evaluated for its in vitro antiproliferative activity against a panel of human tumour cell lines. Compound 3 exhibited the most powerful cytotoxicity against K562 cells, being approximately 100-fold more potent than tiazofurin. This analogue was also active against Jurkat, HT-29 and HeLa malignant cells, with respective IC(50) values being ca. 2-, 27- and 17-fold lower than those observed for tiazofurin. Remarkably, compound 3 did not exhibit any significant cytotoxicity towards normal foetal lung MRC-5 cell line.     

DNA triplex stabilization by a delta-carboline derivative tethered to third strand oligonucleotides

A delta-carboline derivative was covalently coupled to a 7 mer oligonucleotide at its 5'- or 3'-end. The stability of triplexes formed from the conjugates and a double-helical target was studied by UV melting experiments. Compared to the unmodified control triple helices, triplexes with the conjugate exhibit a significantly higher stability. However, the degree of stabilization depends on the particular triplex structure formed.     

Transformations of bioactive peptides in the presence of sugars--characterization and stability studies of the adducts generated via the Maillard reaction

Glycation of biomolecules, such as proteins, peptide hormones, nucleic acids, and lipids, may be a major contributor to the pathological manifestations of aging and diabetes mellitus. These nonenzymatic reactions, also termed the Maillard reaction, alter the biological and chemical properties of biomolecules. In order to investigate the effect of various reducing sugars on the products formed from small bioactive peptides (Tyr-Gly-Gly-Phe-Leu, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OMe, Tyr-Gly-Gly-Phe, and Tyr-Gly-Gly), model systems were prepared with glucose, mannose or galactose. Peptide-sugar mixtures were incubated at 37 or 50 degrees C in phosphate-buffered saline, pH 7.4, or in methanol. The extent of glycation was determined periodically by RP HPLC. All sugar-peptide mixtures generated two different types of glycation products: N-(1-deoxy-ketos-1-yl)-peptide (Amadori compound) and the imidazolidinone compound substituted by sugar pentitol and peptide residue. The amount and distribution of peptide glycation products depended on the structure of the reactants, and increased in both concentration- and time-dependent manner in relation to exposure to sugar. Additionally, the rate of hydrolysis of glucose-derived imidazolidinone compounds, obtained either from leucine-enkephalin (1) or its shorter N-terminal fragments 2 and 3, was determined by incubation at 37 degrees C in human serum. These results revealed that imidazolidinones obtained from glucose and small peptides are almost completely protected from the action of enzymes in serum, the predominant route of degradation being spontaneous hydrolysis to initial sugar and peptide compound.     

Novel cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and thieno[3',2':4,5]thieno- and thieno[2',3':4,5]thieno [2,3-c]quinolones: synthesis, photochemical synthesis, DNA binding, and antitumor evaluation

A series of cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and their 'cyclic' derivatives (quinolones) were synthesized. 'Cyclic' compounds displayed a rather strong and differential antiproliferative effect on various cell lines, while the 'acyclic' amidino-substituted compounds were much more active, but showing mostly non-differential cytotoxicity, whereas cyano-substituted compounds (2a,b) produced a strikingly strong effect selectively on HeLa and Hep-2 cell lines. Antiproliferative activity of 'cyclic' derivatives is very likely caused by intercalation into DNA, while their 'acyclic' analogues use other target(s) and/or mechanisms of action.     

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.     

Influence of sorbitol on protein production and glycosylation and cell wall formation in Trichoderma reesei

Sorbitol is often used at 1 mol/liter as an osmotic stabilizer for cultivation of fungi with a fragile cell wall phenotype. On the other hand, at this concentration sorbitol causes an osmotic stress in fungal cells resulting in intensive production of intracellular glycerol. The highly increased consumption of glucose for glycerol synthesis may lead to changes in processes requiring carbohydrate residues. This study provides new information on the consequences of osmotic stress to the cell wall composition, protein production and glycosylation, and cell morphology of Trichoderma reesei. We observed that high osmolarity conditions enhanced biomass production and strongly limited synthesis of cell wall glucans and chitin. Moreover, in these conditions the amount of secreted protein decreased nearly ten-fold and expression of cbh1 and cbh2 genes coding for cellobiohydrolase I and cellobiohydrolase II, the main secretory proteins in T. reesei, was inhibited resulting in a lack of the proteins in the cell and cultivation medium. The activity of DPM synthase, enzyme engaged in both N- and O-glycosylation pathways, was reduced two-fold, suggesting an overall inhibition of protein glycosylation. However, the two modes of glycosylation were affected divergently: O-glycosylation of secreted proteins decreased in the early stages of growth while N-glycosylation significantly increased in the stationary phase.