Une des difficultés rencontrées lors de l'administration d'agents thérapeutiques au sein du parenchyme cérébral peut être résolue par l'utilisation de nouvelles formes galéniques implantables. Nous montrons dans ce travail la biocompatibilité dans le cerveau de rat des microsphères synthétisées par Biocompatibles facilement injectées par stéréotaxie et chargées en doxorubicine (1mg/ml) (CM-BC1) ou en irinotécan (100mg/ml) (CM-BC2). Nous avons ensuite rapporté l'efficacité des CM-BC2 sur le gliome 9L du rat avec une amélioration significative de la survie par rapport aux groupes non traité ou traité par microsphères blanches. La combinaison des traitements n'améliore pas significativement la survie par rapport à la radiothérapie seule. La récente expansion des nanotechnologies associée aux progrès des méthodes mini-invasives d'administration intracérébrale de médicaments offre l'opportunité d'améliorer ces résultats. Nous avons étudié, toujours sur lemodèle 9L, l'effet radiosensibilisant du paclitaxel, injecté en intratumoral par Convection-Enhanced Delivery (CED) et vectorisé par des nanocapsules lipidiques (LNC) développées par le laboratoire. Une tendance à l'augmentation se profile en faveur des LNC de paclitaxel qui permettent l'obtention de la meilleure médiane de survie (+10 jours par rapport au groupe irradié seul). Bien que les résultats soient non significatifs, l'association d'une chimiothérapie locale à la radiothérapie est intéressante. Il reste maintenant à optimiser les connaissances sur ce vecteur avec notamment possibilité d'adressage subcellulaire et amélioration de la cinétique de libération du principe actif.

Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical.

by Po Chu, Leung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-169). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviation --- p.iii / Abstract --- p.vii / Chinese Abstract --- p.x / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- The Development of Hematopoietic Progenitor Cells --- p.1 / Chapter 1.1.2 --- Hematopoietic Growth Factors --- p.3 / Chapter 1.1.3 --- Transcriptionl Factors Involved in Lineage Commitment of Hematopoietic Progenitor Cells --- p.5 / Chapter 1.2 --- Leukemia --- p.7 / Chapter 1.2.1 --- Occurrence and Classification of Leukemia --- p.7 / Chapter 1.2.2 --- The Pathological Features and Etiology of Leukemia --- p.10 / Chapter 1.2.3 --- The Molecular Basis of Leukemia --- p.13 / Chapter 1.2.4 --- Current Therapeutic Strategies --- p.14 / Chapter 1.2.4.1 --- Conventional Therapies for Leukemia --- p.14 / Chapter 1.2.4.2 --- Induction of Cell Differentiation and Apoptosis for Treatment of Leukemia --- p.16 / Chapter 1.2.5 --- The Use of Murine Myelomonocytic Leukemia WEHI-3B JCS Cells As a Model for the Study of Leukemia Cell Proliferation， Differentiation and Survival --- p.22 / Chapter 1.3 --- Taxol: A Novel Anti-cancer Agent --- p.23 / Chapter 1.3.1 --- Discovery and Action Mechanism --- p.23 / Chapter 1.3.2 --- Metabolism and Toxicity of Taxol --- p.27 / Chapter 1.3.3 --- The Biological Activities of Taxol --- p.28 / Chapter 1.3.4 --- The Anti-tumor Effects of Taxol --- p.30 / Chapter 1.3.5 --- The Effects of Taxol on Leukemia --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.32 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Mice --- p.35 / Chapter 2.1.3 --- "Culture Media,Buffer and Other Solutions" --- p.37 / Chapter 2.1.4 --- Radioisotope and Scintillation Fluid --- p.39 / Chapter 2.1.5 --- Taxol --- p.40 / Chapter 2.1.6 --- Recombinant Cytokines --- p.40 / Chapter 2.1.7 --- Vitamin Analogs --- p.42 / Chapter 2.1.8 --- Various Signal Transduction Pathway Activators and Inhibitors --- p.42 / Chapter 2.1.9 --- Monoclonal Antibodies and Buffers for Flow Cytometry --- p.43 / Chapter 2.1.10 --- Reagents and Chemicals for Gene Expression Study --- p.45 / Chapter 2.1.11 --- Chemical Solutions and Buffers for Western Blot --- p.50 / Chapter 2.1.12 --- Reagents for Colony Assay --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Culture of Leukemia Cell Lines --- p.55 / Chapter 2.2.2 --- Treatment of Leukemia Cells with Various Drugs and Cytokines --- p.55 / Chapter 2.2.3 --- Cell Morphological Study --- p.55 / Chapter 2.2.4 --- Determination of Leukemia Cell Survival and Proliferation --- p.56 / Chapter 2.2.5 --- Colony Assay --- p.56 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.57 / Chapter 2.2.6.1 --- Surface Antigen Immunophenotyping --- p.57 / Chapter 2.2.6.2 --- Assay of Endocytic Activity --- p.58 / Chapter 2.2.6.3 --- Cell Cycle /DNA Content Evaluation --- p.58 / Chapter 2.2.7 --- Gene Expression Study --- p.59 / Chapter 2.2.7.1 --- Preparation of Total Cellular RNA --- p.59 / Chapter 2.2.7.2 --- Reverse Transcription --- p.60 / Chapter 2.2.7.3 --- Polymerase Chain Reaction (PCR) --- p.60 / Chapter 2.2.7.4 --- Agarose Gel Electrophoresis --- p.61 / Chapter 2.2.8 --- DNA Fragmentation Analysis --- p.61 / Chapter 2.2.9 --- Protein Expression Study --- p.62 / Chapter 2.2.9.1 --- Protein Extraction --- p.62 / Chapter 2.2.9.2 --- Quantification of the Protein --- p.62 / Chapter 2.2.9.3 --- Western Blot Analysis --- p.63 / Chapter 2.2.10 --- Statistical Analysis --- p.64 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Effects of Taxol on the Proliferation and Apoptosis of the Murine Myeloid Leukemia Cells --- p.65 / Chapter 3.1.1 --- Growth-Inhibitory Effects of Taxol on Murine Myeloid Leukemia WEHI-3B JCS cells --- p.65 / Chapter 3.1.2 --- Cytotoxic Effects of Taxol on Murine Bone Marrow Cells and Myeloid Leukemia WEHI-3B JCS Cells --- p.69 / Chapter 3.1.3 --- Anti-proliferative Effect of Taxol on Different Leukemia Cell Lines --- p.72 / Chapter 3.1.4 --- Effects of Taxol on the Cell Cycle Kinetics of WEHI-3B JCS Cells --- p.81 / Chapter 3.1.5 --- Induction of DNA Fragmentation of WEHI-3B JCS cells by Taxol --- p.83 / Chapter 3.1.6 --- Effect of Taxol on the Clonogenicity of WEHI-3B JCS Cells In Vitro and Tumorigenicity In Vivo --- p.86 / Chapter 3.2 --- Effects of Taxol on the Induction of Monocytic Cell Differentiation in Murine Myeloid Leukemia Cells --- p.88 / Chapter 3.2.1 --- Morphological Changes in Taxol-Treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.88 / Chapter 3.2.2 --- Surface Antigen Immunophenotyping of Taxol-treated WE HI-3B cells --- p.91 / Chapter 3.2.3 --- Endocytic Activity of Taxol-treated WEHI-3B JCS cells --- p.95 / Chapter 3.3 --- Modulatory Effect of Taxol and Cytokines on the Proliferation of WEHI- 3B JCS Cells --- p.96 / Chapter 3.4 --- Modulatory Effect of Taxol and Physiological Differentiation Inducers on the Proliferation of WEHI-3B JCS cells --- p.103 / Chapter 3.5 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.106 / Chapter 3.6 --- Modulation of Apoptotic Gene Expression in Taxol-treated WEHI-3B JCS cells --- p.113 / Chapter 3.7 --- Modulatory Effects of Taxol on the Protein Expression of WEHI-3B JCS Cells --- p.119 / Chapter Chapter 4: --- Discussion and Conclusions / Chapter 4.1 --- "Effects of Taxol on the Proliferation,Differentiation and Apoptosis of the Murine Myeloid Leukemia Cells" --- p.126 / Chapter 4.2 --- "Modulatory Effects of Taxol, Cytokines and Physiological Differentiation Inducers on the Proliferation of the Myelomonocytic Leukemia WEHI-3B JCS Cells" --- p.132 / Chapter 4.3 --- The Possible Involvement of Protein Kinase C in Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.136 / Chapter 4.4 --- The Modulation of Apoptosis Gene Expression in Taxol-treated WEHI-3B JCS Cells --- p.137 / Chapter 4.5 --- The Modulation of Protein Expression in Taxol-treated WEHI-3B JCS Cells --- p.138 / Chapter 4.6 --- Conclusions and Future Perspectives --- p.139 / References --- p.141

Numerous drugs in the current pharmacopoeia originate from plant sources. Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel. Paclitaxel is mainly derived from the plant genus Taxus and has been widely used in cancer chemotherapy. However, plant cell culture is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. Therefore, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs) from Taxus cuspidata, which possess superior growth properties relative to DDCs. These CMCs have been demonstrated to be a cost effective platform for the sustainable production of paclitaxel. Using 454 sequencing, we determined the transcriptome of T. cuspidata CMCs. Utilizing this transcriptome as a reference, we then employed Solexa digital gene expression profiling to identify transcriptional regulators that were induced by methyl jasmonate, an activator of paclitaxel biosynthesis. This lead to the discovery of 19 putative transcription factors (TFs) belonged to 5 TF families which were further confirmed by associated molecular methods. We aimed to identify which of these 19 regulatory proteins drive the expression of 5 paclitaxel biosynthetic genes by employing yeast one-hybrid analysis and electrophoretic mobility shift assays. Further, the cis-regulatory elements associated with these TFs were identified in the promoter regions of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferese (DBAT), phenylalanine aminomutase (PAM) and 3'-N-debenoyl-2-N-benzoyltransferase (DBTNBT), paclitaxel biosynthetic genes to facilitate the TF-DNA binding studies. Finally, understanding the TF regulatory network underlying paclitaxel biosynthesis can guide the engineering of CMCs to elevate the production of this key pharmaceutical.

Orientador: Ramon Kaneno / Resumo: O câncer de próstata é um dos principais tipos de câncer em todo o mundo, sendo o que causa maior morbidade. Apesar da alta probabilidade de cura nos casos com diagnóstico precoce, essa neoplasia é responsável por mais de 80% dos casos de metástases ósseas e, uma vez detectada, a doença maligna é considerada incurável. Os íons de cálcio são fundamentais da maquinaria celular e sua concentração está intimamente ligada a processos vitais, entre eles a proliferação celular. Visto que as células tumorais são mais sensíveis e dependentes dos níveis de cálcio para manutenção da proliferação, apresentando maior avidez por esse elemento que as células normais, no presente estudo testamos a hipótese de que o revestimento, com cálcio (hidroxiapatita), de nanopartículas magnéticas de ferrita Mn-Zn polimerizadas com quitosana, poderia endereçar o fármaco paclitaxel às células de câncer prostático, levando-as à morte celular. Coerente com nossa hipótese inicial, nossos resultados para as nanopartículas mimetizadas com hidroxiapatita mostraram-se com grande potencial citotóxico para células de câncer de próstata LNCaP, sem, entretanto, afetar pró-osteoblastos murinos da linhagem MC3T3. As nanopartículas contendo apenas paclitaxel (bio-NCP-PTX) promoveram níveis de morte celular muito próximos aos observados com concentração similar de PTX puro, apresentando, portanto, resultado superior ao compósito revestido com Ca (bio-NCP-PTX-APA). Por fim, observamos que o núcleo magnético, a matriz po... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prostate cancer figures among the most frequent types of cancer in the world, being the one with greatest morbidity. Despite the high probability of cure of early diagnosed cases, this neoplasm is responsible for more than 80% of cases of bone metastases and, once achieved the disease is considered incurable. Calcium ions are fundamental to the cell machinery and their concentration is closely linked to vital processes, among them cell proliferation. Since tumor cells are more sensitive and dependent on calcium levels to maintain proliferation, being more avid for this element than normal cells, in the present study we tested the hypothesis that mimicryof magnetic MN-Zn nanoparticles polymerized with chitosan, with calcium (hydroxyapatite), would be able to address paclitaxel to prostate cancer cells, leading to cell death. In agreement with our hypothesis, our results show that hydroxyapatite-mimicked nanoparticles have a high cytotoxic potential to LNCaP prostate cancer cells, with no toxicity to non malignant pro-osteoblast murine cell line. Nanoparticles containing only paclitaxel (bio-NCP-PTX) promoted cell death in a level close to that induced by similar concentration of pure paclitaxel. It indicates that this composite worked better than that covered by Ca (bio-NCP-PTX-APA). Finally, we observed that the magnetic core, with the polymer matrix of chitosan coated or not with hydroxyapatite, does not have toxic effects alone. Thus, these results indicate that these nanop... (Complete abstract click electronic access below) / Mestre

Palliation therapy for dysphagia using esophageal stents is the current treatment of choice for those patients with inoperable esophageal malignancies. However, the stents currently used in the clinical setting, regardless of the type of metal mesh or plastic mesh stents (covered/uncovered), may cause complications, such as tumor ingrowth and stent migration into the stomach. Furthermore, metal mesh stents have limited capacities for loading anti-cancer drugs. To effectively reduce/overcome those complications and enhance the efficacy of drug release, we designed and 3D-printed a tubular, flexible polymer stent with spirals, and then load anti-cancer drug, paclitaxel, on the stent for drug release. Non- spiral 3D-printed tubular and mesh polymer stents served as controls. The self-expansion and anti migration properties, cytotoxicity, drug release profile, and cancer cell inhibition of the 3D-printed stent were fully characterized. Results showed the self-expansion force of the 3D-printed polymer stent with spirals was slightly higher than the stent without spirals. The anti-migration force of the 3D-printed stent with spirals was significantly higher than the anti-migration force of a non-spiral stent. Furthermore, the stent with spirals significantly decreased the migration distance compared to the migration distance of the non-spiral 3D-printed polymer stent. The in vitro cytotoxicity of the new stent was examined through the viability test of human esophagus epithelial cells, and results indicated that the polymer stent does not have any cytotoxicity. The results of in vitro cell viability of esophageal cancer cells further indicated that the paclitaxel in the spiral stent treated esophageal cancer cells much more efficiently than that in the mesh stent. Furthermore, the results of the in vitro drug release profile and drug permeation showed that the dense tubular drug-loaded stent could efficiently be delivered more paclitaxel through the esophageal mucosa/submucosa layers in a unidirectional way than mesh stent that delivered less paclitaxel to the esophageal mucosa/submucosa but more to the lumen. In summary, these results showed that the 3D-printed dense polymer stent with spirals has promising potential to treat esophageal malignancies. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Lung cancer is the leading cause of cancer-related death and the second most diagnosed cancer in the United States. Unfortunately, many patients either do not have any common mutations for which there are already targetable agents, or they eventually become resistant to these compounds. As such, there is a high demand for new, effective methods of treating this disease as well as predicting patient prognosis and potential benefit from chemotherapy. In this work, numerous strategies for treating lung cancer are explored. The first method described here is through the use of a pan-early 2 factor (E2F) inhibitor, HLM006474, which is shown to synergize with paclitaxel in non-small cell lung cancer (NSCLC). Next, we explored the creation and utilization of an E2F signature that is prognostic and predictive of early-stage NSCLC patient benefit from adjuvant chemotherapy (ACT). The third project examined possible targets to enhance sensitivity to cisplatin in NSCLC lacking Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma receptor tyrosine kinase (ALK) fusions (triple-negative), for which cisplatin is one of the few treatment options. These studies led to the identification of a kinase that is overexpressed in NSCLC and whose knockdown sensitizes cells to platinum agents.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-104). Also available in print.

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