Yes / The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose–response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell–cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA. / The full-text of this article was released for public view at the end of the publisher embargo on 2 Feb 2016.

No description available.

O glioblastoma multiforme (GBM) é um dos gliomas mais comuns, classificado como um glioma de grau IV (Organização Mundial da Saúde - OMS) e notoriamente difícil de ser tratado. O tratamento recomendado consiste na ressecção cirúrgica seguida de radio e quimioterapia, e a sobrevida média dos pacientes é de apenas 12 meses após o diagnóstico. Portanto, novas terapias que focam a redução do volume do tumor e o aumento da morte das células tumorais são urgentemente necessárias. Estudos pré-clínicos sugerem que a inibição de COX-1 e 2 com Anti-inflamatórios não esteroidais (AINEs), como o Ibuprofeno (IBP), inibiram significantemente a proliferação e a migração celular em diferentes tumores. Assim, o presente estudo teve como objetivo avaliar in vitro o efeito da inibição de COX-1 e COX-2 através do tratamento com IBP, e também com inibidores específicos como SC-560 e NS-398 respectivamente, na migração e na proliferação da linhagem celular de glioma humano U251-MG. O presente estudo demonstrou através de diversos ensaios que a inibição de COX-1 e COX-2 através do IBP, do SC-560 e do NS-398 inibiu significativamente a migração e a proliferação celular da linhagem celular U251-MG. Dessa maneira, concluímos que a PGE2 está envolvida na migração e proliferação celular das células desta linhagem celular. / Glioblastoma Multiforme (GBM) is the most common glioma, classified as grade IV (World Health Organization WHO) and notoriously difficult to treat. The recommended treatment consists of surgery followed by radiotherapy and chemotherapy, and the median survival is ten to twelve months. Preclinical studies suggest that inhibiton of cyclooxygenase-2 by treatment with non-steroidal anti-inflammatory drugs, such as ibuprofen, significantly blocked the proliferation of different tumors including gliomas. Thus this study aimed to evaluate the migration of glioma cell line U251-MG after inhibition of cyclooxygenases 1 and 2 by treatment with ibuprofen (IBP), and specifics inhibitors such as SC-560 for COX-1 and NS-398 for COX-2. The present study demonstrated by various assays where inhibition of COX-1 and COX-2 by IBP, SC-560 and NS-398, significantly inhibited migration and cell proliferation of U251-MG cell line. Thus, we conclude that PGE2 is involved in this cell line migration and cell proliferation.

O glioblastoma multiforme (GBM) é um dos gliomas mais comuns, classificado como um glioma de grau IV (Organização Mundial da Saúde - OMS) e notoriamente difícil de ser tratado. O tratamento recomendado consiste na ressecção cirúrgica seguida de radio e quimioterapia, e a sobrevida média dos pacientes é de apenas 12 meses após o diagnóstico. Portanto, novas terapias que focam a redução do volume do tumor e o aumento da morte das células tumorais são urgentemente necessárias. Estudos pré-clínicos sugerem que a inibição de COX-1 e 2 com Anti-inflamatórios não esteroidais (AINEs), como o Ibuprofeno (IBP), inibiram significantemente a proliferação e a migração celular em diferentes tumores. Assim, o presente estudo teve como objetivo avaliar in vitro o efeito da inibição de COX-1 e COX-2 através do tratamento com IBP, e também com inibidores específicos como SC-560 e NS-398 respectivamente, na migração e na proliferação da linhagem celular de glioma humano U251-MG. O presente estudo demonstrou através de diversos ensaios que a inibição de COX-1 e COX-2 através do IBP, do SC-560 e do NS-398 inibiu significativamente a migração e a proliferação celular da linhagem celular U251-MG. Dessa maneira, concluímos que a PGE2 está envolvida na migração e proliferação celular das células desta linhagem celular. / Glioblastoma Multiforme (GBM) is the most common glioma, classified as grade IV (World Health Organization WHO) and notoriously difficult to treat. The recommended treatment consists of surgery followed by radiotherapy and chemotherapy, and the median survival is ten to twelve months. Preclinical studies suggest that inhibiton of cyclooxygenase-2 by treatment with non-steroidal anti-inflammatory drugs, such as ibuprofen, significantly blocked the proliferation of different tumors including gliomas. Thus this study aimed to evaluate the migration of glioma cell line U251-MG after inhibition of cyclooxygenases 1 and 2 by treatment with ibuprofen (IBP), and specifics inhibitors such as SC-560 for COX-1 and NS-398 for COX-2. The present study demonstrated by various assays where inhibition of COX-1 and COX-2 by IBP, SC-560 and NS-398, significantly inhibited migration and cell proliferation of U251-MG cell line. Thus, we conclude that PGE2 is involved in this cell line migration and cell proliferation.

Tyrimo metodai: Ląstelių sėjimo metodika; ląstelių tankio nustatymas; ląstelių gyvybingumo nustatymas MTT testu; neuronų žūties įvertinimas fluorescencinės mikroskopijos metodu; viduląstelinių RS kiekio įvertinimas; ekstraląstelinių RS kiekio įvertinimas; statistinė analizė. Tyrimo tikslas: Ištirti įvairių koncentracijų lektinų, išskirtų iš Phaseolus vulgaris L., antioksidacinį/ prooksidacinį poveikį ir įtaką C6 ląstelių gyvybingumui. Tyrimo uždaviniai: 1. Atlikti literatūros duomenų analizę apie lektinų, išskirtų iš Phaseolus vulgaris L., biologinį poveikį; 2. Nustatyti įvairių koncentracijų lektinų, išskirtų iš Phaseolus vulgaris L., antioksidacinį/prooksidacinį aktyvumą; 3. Nustatyti įvairių koncentracijų lektinų poveikį C6 ląstelių kultūros gyvybingumui. Tyrimų rezultatai: 1. Buvo matuojami vandenilio peroksido pokyčiai esant 1 - 10 µg lektino koncentracijoms ir nustatyta, kad skirtingų koncentracijų lektinas neveikia neutralizuojančiai vandenilio peroksido kiekio. 2. Atlikti vandenilio peroksido matavimai DMEM terpėje su 1 - 10 µg lektino koncentracijoms parodė, kad lektinas, priklausomai nuo jo koncentracijos, negeneruoja laisvųjų radikalų. 3. Buvo matuojamas glioblastomos C6 ląstelių gyvybingumas su MTT po 24 valandų ir nustatyta, kad didėjant lektino koncentracijai ląstelių gyvybingumas sparčiai mažėjo: 5 µg koncentracijos lektinas gyvybingumą sumažino beveik 2 kartus (iki 57,2111.16%), o 10 µg koncentracijos lektinas ląstelių gyvybingumą sumažino beveik 13... [toliau žr. visą tekstą] / Methods: The cell cultivation method; measurement of cell density; determination of cell viability with MTT assay; assessment of neuronal death with fluorescent microscopy; measurement of intracellular RS; measurement of extracellular RS; statistical analysis. The aim of research: To investigate antioxidative/prooxidative effects of different concentrations of the lectin, isolated from Phaseolus vulgaris L., and influence on the viability of the C6 cells. Goals of the study: 1. To perform an analysis of the literature on the biological effects of lectin, isolated from Phaseolus vulgaris L.; 2. To determine antioxidative/prooxidative activity of different concentrations of the lectin, isolated from Phaseolus vulgaris L.; 3. To determine effect on viability of the C6 cell culture using different concentrations. Results: 1. Concentrations of hydrogen peroxide were measured with 1 - 10 µg concentrations of lectin and it was found, that different concentrations of lectin did not neutralize hydrogen peroxide. 2. Measurements of hydrogen peroxide in DMEM medium with 1-10 mg concentrations of lectin showed, that the lectin, depending on it's concentration, did not generate free radicals. 3. Viability of cells was measured in C6 glioblastoma cells with MTT after 24 hours and it was found, that when concentration of lectin increased, viability of cells rapidly decreased: 5 µg concentration of lectin reduced the viability of almost 2 times ( up to 57.21% ± 11.16% ) and at 10 µg... [to full text]

Das Glioblastom ist mit 70 % aller Gliome der häufigste humane Hirntumor mit sehr ungünstiger Prognose. Es konnte gezeigt werden, dass das natürlich vorkommende Dipeptid Carnosin (β-Alanyl-L-histidin) die Proliferation von Glioblastomzellen inhibiert. Diese Wirkung des Carnosins konnte ebenfalls in vivo nachgewiesen werden. Da Carnosin auch einen Einfluss auf den ATP-Haushalt der Glioblastomzellen besitzt, war das Ziel dieser Arbeit einen Wirkungsort von Carnosin zu identifizieren, womit die ATP mindernden und proliferationshemmenden Eigenschaften erklärt werden können. Es wurde untersucht, ob Carnosin den Energiemetabolismus der Glioblastome beeinflusst. Dabei konnte mithilfe zellbiochemischer Methoden gezeigt werden, dass die untersuchten Zelllinien nicht von der Energieversorgung durch die mitochondriale oxidative Phosphorylierung abhängen, da sich weder Hemmung (KCN) noch Entkopplung (DNP) der Elektronentransportkette auf den zellulären ATP-Gehalt auswirkten. Carnosin hingegen verringerte den ATP-Spiegel dieser Zellen. Die Hemmung der Glykolyse durch Oxamat (LDH-Hemmung), bewirkte einen starken Abfall des intrazellulären ATP-Spiegels, worauf Carnosin keinen zusätzlichen Effekt mehr besaß. Carnosin konnte eine Wirkung auf die glykolytische ATP-Synthese zugesprochen werden. Da ein direkter, molekularer Wirkungsort auf diesem Weg nicht identifiziert werden konnte, wurde parallel untersucht, ob sich über Proteomanalysen der Glioblastomzelllinie T98G ein Wirkungsort, bzw. -mechanismus bestimmen lässt. Anhand der Methode der zweidimensionalen Gelelektrophorese (2D-GE) konnten 31 signifikant differenziell exprimierte Proteine detektiert werden, von denen 6 Proteine (VBP-1, OLA-1, TALDO 1, UROD, BAG-2, GRPEL1) über MALDI-TOF-Analysen identifiziert wurden. In Western-Blot-Analysen konnte ein Protein (VBP-1), neben T98G, auch in primären Glioblastomzelllinien als differenziell exprimiert nachgewiesen werden. Anhand der zellbiologischen und proteinbiochemischen Untersuchungen konnte einerseits eine Verbindung des Carnosins zum HIF1α-Signalweg und andererseits zur generellen posttranslationalen Peptidprozessierung hergestellt werden. Der direkte Nachweis eines Einflusses von Carnosin auf HIF1α wurde aber bisher nicht erbracht.

Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor that mainly affects adults. GBM displays remarkable intra- and inter-tumoral heterogeneity and contains a subpopulation of cells named glioma stem cells that is believed to be responsible for tumor maintenance, progression and recurrence. We have established and characterized a biobank of 48 cell lines derived from GBM patients. The cells were explanted and maintained as adherent cultures in serum-free, defined neural stem cell medium. These GBM cells (GCs) displayed NSC marker expression in vitro, had orthotopic tumor initiating capability in vivo, harboured genomic alterations characteristic of GBM and represented all four TCGA molecular subtypes. Our newly established biobank is also connected with a database (www.hgcc.se) that provides all molecular and clinical data. This resource provides a valuable platform of valid in vitro and in vivo models for basic GBM research and drug discovery. By using RCAS/tv-a mouse models for glioma, we found that GBMs originating from a putative NSC origin caused more tumorigenic GCs that had higher self-renewal abilities than those originating from putative glial precursor cell origin. By transcriptome analysis a mouse cell origin (MCO) gene signature was generated to cluster human GCs and GBM tissue samples and a functional relationship between the differentiation state of the initially transformed cell and the phenotype of GCs was discovered, which provides the basis for a new predictive MCO-based patient classification. LGR5 was found to be highly expressed in the most malignant mouse GC lines of putative NSC origin and also enriched in proneural GBMs characterized by PDGFRA alterations and OLIG2 up-regulation. By overexpressing or depleting LGR5 we discovered that high LGR5 expression in proneural GC lines increased the tumorigenicity, self-renewal and invasive capacities of the cells and could potentiate WNT signalling through its ligand RSPO1. Through transcriptome analysis we identified the candidate genes CCND2, PDGFRA, OLIG2, DKK1 that were found to be regulated by LGR5. In the last study, we found that mouse OPCs could initiate both astrocytic and oligdendroglial gliomas, which indicated that oncogenic signalling is dominant to cell of origin in affecting the histology of gliomas.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour in adults. The employment of current standard of care management strategy, that is combining maximum but safe surgical resection, and concomitant chemoradiotherapy, only achieves very modest survival benefits. Antiangiogenesis is a widely studied therapeutic strategy, which restricts the tumour growth by cutting off blood supplement. Although several antiangiogenic agents are now under clinicaland preclinical trials, bevacizumab is still the only one that has been proven to be effective in the treatment of recurrent GBM. However, the clinical use of bevacizumab has encountered the emergence of drug resistance. Its therapeutic benefit is considered limited because of its single pathway targeting. Many researchers believe that the use of broad spectrum angiogenesis inhibitors may leadto better clinical outcomes by overcoming the shortcomings of bevacizumab. / Vastatin, the globular non-collagenous 1 (NC1) domain of collagen VIIIα1, was initially proved to inhibit the proliferation and migration of bovine aortic endothelial cells. Although vastatin is similar in origin to other collagen-derived antiangiogenic factors (CDAFs), its antiangiogenic capability in treatment of cancers has not been studied systematically. Our team members previously found that vastatin wasa safe and effective antiangiogenic therapeutic and a potential biomarker for liver cancer. In this thesis, I tried to explore the therapeutic potential of vastatin in treatment of GBM. / Using a recombinant adeno-associated virus mediated gene therapy, the antiangiogenic potential of vastatin was first confirmed in vitro that it inhibited proliferation, migration and tube formation of murine microvascular endothelial cells (MECs). These effects were further confirmed using another gene vector (H1) which was subsequently employed for the in vivo studies. H1 is a nanopolymer gene vector has high affinity with the folate receptors on tumour cells. Transfection ofH1/vastatin reduced MEC proliferation in a U87/MEC co-culture system, suggesting a paracrine inhibition. Mechanism studies showed that vastatin caused a wide range of changes in the global gene transcription level in MECs, indicating a broad spectrum of action. / Following the establishment of an orthotopic murine GBM model, the H1/DNA polyplexes were injected directly to the tumour area. Treatment induced a significant increase in intracranial mRNA level of the therapeutic gene. Both vastatin and endostatin, a positive control, prolonged the survivals of GBM bearing mice. Immunostaning showed that vastatin decreased microvessel density in the outer layer of the tumour, while decreased cell density and caused abnormal vessel structures inthe centre. No synergistic effect was observed when GBM was treated with the combination of H1/vastatin and temozolomide (TMZ) in this model. / Finally, the therapeutic effect of vastatin on a TMZ resistant model was studied. GBM cells with acquired TMZ resistance (ATR) were established by chronic exposure of U87 cells to TMZ. Animals grafted with the U87-ATR cells were proved to be tolerant of TMZ treatment. H1/vastatin injection significantly prolonged the survival in this model. More interestingly, H1/vastatin also resensitized these animals to TMZ treatment. Stem cell related drug resistance was supposed to be disturbed in this process. / In conclusion, the present study has demonstrated for the first time that vastatin, a broad spectrum endogenous angiogenesis inhibitor, is of therapeutic benefit in a murine orthotopic GBM model. Vastatin’s capability to reverse TMZ resistance highlights an important area for further research. / 胶质母细胞瘤(GBM)是成人最常见的恶性原发性脑肿瘤。目前的治疗手段包括了手术切除和放化疗，但是效果仍不能让人满意。与传统的化疗药不同，抗血管生成药物能通过抑制肿瘤内新血管的形成，切断血流供给，达到限制肿瘤生长的目标。贝伐单抗（Bevacizumab)是目前唯一获得批准用于临床GBM治疗的抗血管生成药物。然而Bevacizumab在临床应用中必须面对耐药性产生的问题， 而且因为Bevacizumab只单一性地阻断血管内皮生长因子相关的通路，所以它的治疗效果也受到了一定程度的限制，让肿瘤可以选择替代性的通路来获得新生血管。因此一些研究人员认为，改用多靶点或者广谱的抗血管生成药物，治疗效果应该会更好。 / Vastatin是VIII型胶原蛋白α1链上的球状非胶原裂解片断。人体内这一类的片段多被证明了具有抗血管生成的功能，它们统称为“源自胶原蛋白的抗血管生成因子”。Vastatin具有抑制牛主动脉内皮细胞增殖和迁移的作用，然而它在抗肿瘤血管生成方面的作用却没有被系统地研究过。我们之前的实验曾经发现Vastatin对肝癌模型中的血管生成具有明显的抑制效果，而本论文将对Vastatin是否同样具有治疗GBM的作用展开研究。 / 在体外，我们首先证明了重组腺相关病毒（rAAV）介导的Vastatin基因治疗能有效抑制MEC的增殖和迁移，并阻止其形成管状结构。我们同时也测试了另一种基因载体H1，以方便后续动物实验的开展。H1是一种纳米聚合物，对肿瘤细胞表面高表达的叶酸受体有高亲和力。H1 介导的Vastatin 基因治疗对肿瘤细胞和MEC都没有直接的作用，但在两种细胞的共培养体系中，Vastatin可以通过旁分泌的方式来抑制MEC的增殖。对机制的研究发现，Vastatin使MEC内基因转录的水平发生了大范围多通路的改变，说明了它的作用具有一定的广谱性。 / 实验进一步研究了Vastatin在小鼠原位GBM 模型中的作用。将H1/DNA 复合物直接注入瘤区可以明显提高颅内相应基因的转录水平。Vastatin和作为阳性对照的Endostatin都能有效地延长GBM小鼠的生存期。免疫组织化学的结果显示Vastatin 能降低肿瘤内部的微血管密度，并诱导组织坏死。这与之前报道过的Endostatin的作用相似。在同一模型上，我们还测试了Vastatin和Temozolomide(TMZ)结合给药的效果，但并没有了现明显的协同作用。 / 实验最后研究了Vastatin在TMZ耐药模型中的治疗效果。通过将U87细胞长期浸泡中含有TMZ的培养基中，我们成功地筛选出了具有TMZ耐药性的GBM细胞。用这些细胞建立的小鼠GBM模型对TMZ的作用不敏感。实验表明，H1/Vastatin基因疗法不仅能够明显延长模型小鼠的生存期，还可以逆转耐药性，使TMZ重新发挥作用。我们推测干细胞相关的耐药性的产生和维持可能在这个过程中受到了影响。 / 上述研究第一次阐明了Vastatin对GBM的治疗效果。Vastatin具有广谱的抗血管特性，能够通过作用于MEC抑制肿瘤内部新血管的生成。Vastatin不仅本身具有治疗作用，还能逆转动物模型对化疗药物的耐受性，因些具有很高的研究价值。相信对Vastatin更一步的探索不但可以拓宽我们对抗血管生成药物的理解，也可能意味着一个新的研究领域的出现。 / Li, Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 102-110). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / Li, Yi. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Glioblastoma multiforme (GBM) is an aggressive Grade IV astrocytoma with a poor survival rate. This is largely due to the GBM tumor cells migrating away from the primary tumor site along white matter tracts and blood vessels leading to secondary tumor sites. It is unknown whether the microenvironment nanotopography influences the biomechanical properties of the tumor cells. Although these tumor cells have an innate propensity to migrate, we believe that the nanotopography changes the biomechanical properties to enhance the migratory phenotype. To study this, we used an in vitro polycaprolactone aligned nanofiber film that mimics the nanotopography of the white matter tracts and blood vessels to investigate the mechanical properties of the GBM tumor cells. Our data demonstrate that the cytoskeletal stiffness, traction force, and focal adhesion area are inherently lower in invasive GBM tumor cells compared to healthy astrocytes. Moreover, the tumor cytoskeletal stiffness was significantly reduced when cultured on the aligned nanofiber films compared to smooth and randomly aligned nanofibers films. Analysis of gene expression also showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. In addition, cell cycle analysis exhibited a reduced proliferative state on aligned nanofibers, highlighting the dichotomy between proliferation and migration observed in GBM. Finally, focal adhesions of tumor cells were larger and more elliptical when grown on the aligned fibers, suggesting a more migratory state. Therefore, our data demonstrate that the invasive potential is elevated when the tumor cells are cultured on an aligned nanotopography. This in vitro model can further be used to identify the GBM tumor cells’ response in a mimetic in vivo tumor microenvironment and elucidate how the aligned nanotopography transduces into altered gene and protein expression, thus providing a mechanism to target to inhibit the enhanced migratory behavior observed in these cells.