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1
Zha, Beth Shoshana. "HIV Protease Inhibitors Trigger Lipid Metabolism Dysregulation Through Endoplasmic Reticulum Stress and Autophagy." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/273.
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HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART). HIV PIs are extremely effective at suppressing viral load, but have been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease. Recent studies indicate that activation of endoplasmic reticulum (ER) stress is an important cellular mechanism underlying HIV PI-induced dysregulation of lipid metabolism. However, the exact role of ER stress in HIV PI-associated lipodystrophy and dyslipidemia remains to be identified. Hepatocytes and adipocytes are important players in regulating lipid metabolism and the inflammatory state. Dysfunction of these two cell types is closely linked to various metabolic diseases. In this dissertation research, we aimed to define the role of activation of ER stress in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes and further identifty the potential molecular mechanisms. Both cultured and primary mouse adipocytes and hepatocytes were used to examine the effect of individual HIV PIs on ER stress activation and lipid metabolism. The results indicated that HIV PIs differentially activate ER stress through depletion of ER calcium stores, activating the unfolded protein response (UPR). UPR activation further lead to an alteration of cellular differentiation through downstream transcription factor CHOP. At the same time, HIV PIs also altered adipogenesis via differential regulation of the adipogenic transcription factor PPARγ. HIV PI-induced ER stress was closely linked to dysregulation of autophagy activation through CHOP, and upstream ATF-4, signaling pathways. In hepatocytes, the integrase inhibitor raltegravir abrogated HIV PI-induced lipid accumulation by inhibiting ER stress activation and dysregulation of autophagy pathway. Our studies suggest that both ER stress and autophagy are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV HAART-treated patients.
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Khan, Omar Ali. "HR23B, a biomarker for HDAC inhibitors." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:9cd76c0b-e70e-43f7-a92d-a99f403a077e.
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As our understanding of cancer biology increases and novel therapies are developed, an increasing number of predictive biomarkers are becoming clinically available. Aberrant acetylation has been strongly linked to tumourigenesis and the modulation of acetylation through targeting histone deacetylase (HDAC) has led to the introduction of many HDAC inhibitors. To date, two have had regulatory approval for the treatment of cutaneous T cell lymphoma (CTCL). Modifications in chromatin control underpin the mechanism of action of HDAC inhibitors. A genome wide loss-of-function screen identified HR23B as a gene that governs sensitivity to HDAC inhibitors. HR23B shuttles ubiquitinated cargo proteins to the proteasome and elevated levels may contribute to cell death mediated by this pathway. It also governs cell sensitivity to drugs that act directly on the proteasome. HDAC inhibitors influence proteasome activity and there may be a synergistic interaction with proteasome inhibitors. HR23B and HDAC6 interact and HDAC6 may be a negative regulator of apoptosis and a positive regulator of autophagy and through its ability to down-regulate HR23B, may impact on the cellular outcome of HDAC inhibitor treatment. Expression of HR23B has been correlated with clinical response to HDAC inhibitors in a retrospective analysis of CTCL patients. The tissue expression of HR23B and the autophagy marker LC3 has been investigated and there may be a reciprocal relationship in their expression in some tumours which may provide prognostic information and patients with low HR23B expression but high levels of autophagy appear to have a particularly poor prognosis. Well designed, biomarker-driven prospective clinical trials are needed to clarify the predictive and prognostic roles of HR23B.
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Martin, Mackenzie. "Targeting Tau Degradation by Small Molecule Inhibitors for Treatment of Tauopathies." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6314.
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Tauopathies are neurodegenerative diseases that affect millions of people around the world. Tauopathies include more than 20 neurodegenerative diseases. Some of the most common tauopathies are Alzheimer’s disease (AD), frontotemporal dementia (FTD), chronic traumatic encephalopathy (CTE), Pick’s disease, corticobasal degeneration, progressive supranuclear palsy (PSP), agyrophillic grain disease, and amyotrophic lateral sclerosis (ALS). These diseases can cause significant memory loss, behavioral changes, motor deficits and speech impairments. Tauopathies stem from accumulation of the microtubule associated protein tau (MAPT). Tau stabilizes microtubules and helps with axonal transport. In a disease state tau becomes hyperphosphorylated and truncated leading to its aggregation. More recently tau has been shown to propagate from cell to cell potentially acting as a signaling molecule that contributes to disease progression. In addition during disease, tau mislocalizes to dendrites leading to synaptic dysfunction. This mislocalization may also lead to subsequent neurodegeneration.
Today, many strategies have been implemented to treat tauopathies. Some of these strategies include kinase inhibitors, immunotherapy, tau aggregation inhibitors, and microtubule-stabilizing compounds. However none these strategies have been effective in stopping tau pathology nor do they address tau degradation pathways. Therefore we hypothesized that utilizing small molecules that target degradation pathways such as autophagy or proteasomal degradation would improve clearance of aberrant tau.
We previously showed that a natural product (+)-aR,11S-myricanol (1) from Myrica cerifica (bayberry/southern wax myrtle) root bark reduced levels of tau. In this study we discovered that 1 is composed of two enantiomers and two possible atropisomers. We found that one enantiomer (-)-aS,11R-myricanol (3) was responsible for the anti-tau activity of 1 in multiple models of tauopathy. We also found that 3 selectively targets and lowers specific tau species. To better understand how these tau species were being reduced we took a non-biased approach and subjected 3 treated samples to stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry (MS) proteomic analysis. We found that autophagy pathways were most affected by 3 and that 3 was predicted to mimic the drug rapamycin, a well-established macroautophagy activator. In addition we confirmed our MS findings by simultaneously giving 3 treated cells an autophagy inhibitor which blocked 3’s tau reductions. Moreover we created a tetralin derivative of 1, 13, that produced the same effects on tau as 3 but did not rely upon stereochemistry for its activity. This work supports targeting the autophagy degradation pathway as a viable approach to improving aberrant tau accumulation.
In order to further support our hypothesis, we collected and screened several known heat shock protein 70 (Hsp70) inhibitors and tau aggregation inhibitors for cellular anti-tau activity. While it is known that Hsp70 inhibition facilitates tau clearance through proteasomal degradation, it is not known what role tau aggregation inhibition plays in the cellular degradation of tau. Moreover understanding which inhibitory activity contributes most to tau degradation would lead to the creation of better drug scaffolds. In this study, we found that several Hsp70 inhibitors from different scaffold backbones had varying effects on tau degradation. The rhodacyanine and phenothiazine compounds were most effective at lowering cellular tau while the adenosine analog, sulfonamide, dihyropyrimidine, piperidine-3-carboxamide, phenoxy-N-arylacetamide, and flavonol, were not as effective. We also examined the effects of several tau aggregation inhibitor scaffolds such as the carbocyanine, oleuropein, anthraquinone, aminothienopyridazine, hydroxytyrosol and rhodanine on tau expression reduction. We found that none had effective tau reductions except the carbocyanine. However when we performed a lactate dehydrogenase (LDH) assay, carbocyanine was shown to be extremely toxic. These results lead us to further investigate if the tau expression reducing Hsp70 inhibitors had anti-tau aggregation activity and if the tau aggregation inhibitors had any Hsp70 inhibitory activity. We discovered that many of the Hsp70 inhibitors also had anti-tau aggregation activity while none of the aggregation inhibitors had Hsp70 inhibitory activity. We found a positive correlation between tau expression reductions and anti-tau aggregation activity for the Hsp70 inhibitors. Our work demonstrates that both Hsp70 activity and tau aggregation in vitro best predicts anti-tau activity of small molecules. Also these dual acting Hsp70 inhibitors support our hypothesis that targeting the degradation pathways can improve tau clearance.
Overall, this work indicates the importance of targeting degradation pathways to improve tau clearance. Utilizing small molecules that have dual activities against tau could prove beneficial as a novel therapeutic approach to treat tauopathies. In addition using small molecules that target different degradation pathways simultaneously could be another viable therapeutic strategy for treatment of tauopathies.
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Timme, Cindy R. "Drug Resistance Mechanisms to Gamma-secretase Inhibitors in Human Colon Cancer Cells." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4954.
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Colorectal cancer is the third leading cause of cancer-related mortality. Much progress has been achieved in combating this disease with surgical resection and chemotherapy in combination with targeted drugs. However, most metastatic patients develop drug resistance so new modalities of treatment are needed.
Notch signaling plays a vital role in intestinal homeostasis, self-renewal, and cell fate decisions during post-development and is activated in colorectal adenocarcinomas. Under debate is its role in carcinomas and metastatic disease. In theory, blocking Notch activation using gamma-secretase inhibitors (GSIs) may show efficacy alone or in combination with chemotherapy in the treatment of colon cancer.
In Chapter Three, we tested the capacity for GSIs to synergize with oxaliplatin in colon cancer cell lines and evaluated the underlying molecular mechanisms. GSI alone had no effect on colon cancer cell lines. Surprisingly, we show that GSIs blocked oxaliplatin-induced apoptosis through increased protein levels of the anti-apoptotic Bcl-2 proteins Mcl-1 and/or Bcl-xL. Restoration of apoptosis was achieved by blocking Mcl-1 and/or Bcl-xL with obatoclax (an anti-apoptotic Bcl-2 agonist) or siRNA. An unexpected result was the induction of cell death with the combination of GSI and obatoclax.
In Chapter Four, we examined the mechanism of GSI + obatoclax-mediated cell death. We found that apoptosis played a minimal role. Rather, we identified blockage of cytoprotective autophagy played a causative role. Interestingly, we also saw autophagy induction in GSI-treated cells, which could explain the insensitivity of colon cancer cells to GSI. When autophagy was blocked in GSI-treated cells, cells became sensitive to GSI and cell death was elicited. The mechanism by which induction of autophagy occurs in GSI- treated cells is an area for further research.
Overall, our work questions the validity of the use of GSIs in the treatment of colorectal cancers. We show that GSIs may block apoptosis and induce cytoprotective autophagy simultaneously, resulting in increased drug resistance and cellular survival. Whether these two cellular survival processes occurs in patients needs to be examined before GSIs can be utilized in a clinical setting. If so, these two hurdles must be overcome.
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Robke, Lucas [Verfasser], Herbert [Akademischer Betreuer] Waldmann, and Martin [Gutachter] Engelhard. "Discovery and target identification of small molecule autophagy inhibitors / Lucas Robke ; Gutachter: Martin Engelhard ; Betreuer: Herbert Waldmann." Dortmund : Universitätsbibliothek Dortmund, 2017. http://d-nb.info/1139892592/34.
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Rummelt, Marjorie A. [Verfasser], Herbert [Akademischer Betreuer] Waldmann, and Martin [Gutachter] Engelhard. "Identification and biological characterization of indoline-based autophagy inhibitors / Marjorie A. Rummelt ; Gutachter: Martin Engelhard ; Betreuer: Herbert Waldmann." Dortmund : Universitätsbibliothek Dortmund, 2017. http://d-nb.info/113989255X/34.
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Peng, Luo-Gen [Verfasser]. "Urokinase-type plasminogen activator receptor contributes to chemosensitivity and epithelial-to-mesenchymal transition in PDAC : uPAR and p38 regulate autophagy dependent gemcitabine resistance in AsPC1: autophagy inhibitors and gemcitabine as a potential combined therapy for a subgroup of pancreastic cancers / Luogen Peng." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1221802313/34.
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Peng, Luogen [Verfasser]. "Urokinase-type plasminogen activator receptor contributes to chemosensitivity and epithelial-to-mesenchymal transition in PDAC : uPAR and p38 regulate autophagy dependent gemcitabine resistance in AsPC1: autophagy inhibitors and gemcitabine as a potential combined therapy for a subgroup of pancreastic cancers / Luogen Peng." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1221802313/34.
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Garivet, Guillaume [Verfasser], Herbert [Akademischer Betreuer] Waldmann, and Alfred [Gutachter] Wittinghofer. "Small molecule inhibition of lipidated proteins/cargo interaction and synthesis of a cinchona alkaloid-derived library as potent autophagy inhibitors / Guillaume Garivet ; Gutachter: Alfred Wittinghofer ; Betreuer: Herbert Waldmann." Dortmund : Universitätsbibliothek Dortmund, 2017. http://d-nb.info/1149920424/34.
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Meza, Daniel. "Sphingosine Kinase 1 Inhibitor, A Novel Inducer of Autophagy." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1871.
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Autophagy is the process of “cell self-eating” which has been implicated both in cell survival and cell death. Sphingosine kinase 1 (SphK1) regulates the intracellular balance between ceramide and sphingosine, bioactive lipids associated with cell death, and sphingosine-1-phosphate (S1P), whose actions are associated with survival and proliferation. Previous studies have implicated upregulation of SphK1 in the induction of autophagy. In this study, SK1-I, a SphK1 specific competitive inhibitor, induced autophagy in a concentration and time dependent manner in HCT116 colorectal carcinoma cells. This autophagic response was observed to be more intense in wild type p53 expressing HCT116 cells than in p53 null cells and ultimately led to non-apoptotic death in wild type and apoptotic death in p53 null cells. In agreement, cell death in wild type cells was not accompanied by cleavage of polyADP ribose polymerase, a hallmark of apoptosis. Knockdown of Beclin 1 demonstrated that it and its binding partners do not have a significant role in the induction of autophagy in response to SK1-I treatment. Similarly, mTORC1 signaling was not observed. In contrast, SK1-I markedly decreased Akt phosphorylation. However, this might not be the sole factor important for SK1-I induced autophagy, as pharmacological inhibition of Akt only led to a comparatively weak autophagic response. Indeed, phosphorylation of the endoplasmic reticulum (ER) stress marker eIF2 α, was greatly reduced, suggesting that an ER mediated mechanism also contributes to SK1-I induced autophagy. Thus, SK1-I induced autophagy was likely triggered by ER stress signaling and led to non-apoptotic cell death in the more highly autophagic wild type 53 expressing cells. These results suggest that an isotype specific SphK1 inhibitor might be a useful adjunct for the treatment of cancer or other diseases in which enhancement of cytotoxicity or autophagy is desirable.
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Sahin, Katherine B. "Evaluation of cell division cycle associated protein 3 (CDCA3) as a novel prognostic/therapeutic target for EGFR-mutant non-small cell lung cancer." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/231468/1/Katherine_Sahin_Thesis.pdf.
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This thesis defined a unique role for the protein cell division cycle associated protein-3 (CDCA3) in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). This thesis has established an association between the levels of CDCA3 expression and the tumour response to tyrosine kinase inhibitors (TKI), which are the front-line therapy for EGFR-mutant NSCLC. In this disease, CDCA3 functions to modulate cellular growth pathways to impact sensitivity towards TKI therapy. Future work might enable development of a clinical stratification tool to discern TKI responsive from non-responsive EGFR-mutant NSCLC tumours.
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Drullion, Claire. "Réponse et résistance aux inhibiteurs de tyrosine kinases dans le modèle de la LMC : identification et régulation des morts cellulaires." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21872/document.
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La leucémie myéloïde chronique (LMC) est un syndrome myéloprolifératif lié à l’acquisition d’une anomalie chromosomique t(9;22) conduisant à l’expression d’une protéine de fusion p210 Bcr-Abl dont l’activité tyrosine kinase dérégulée est nécessaire et suffisante pour engendrer la maladie.Cette pathologie bénéficie depuis 2002 d’une avancée thérapeutique : les inhibiteurs de tyrosine kinase (ITK). Cette thérapeutique dite ciblée, dont le chef de file est l’imatinib, est très efficace puisque 80% des patients entre en rémission. Malheureusement, 20% des patients traités développent des résistances primaires ou secondaires, dépendantes ou non de l’oncogène Bcr-Abl dont certaines ont été caractérisées. A ce titre, la LMC est devenue un modèle d’étude à la fois des mécanismes oncogéniques mais aussi des résistances.La résistance aux ITK dans la LMC peut être considérée sur deux plans. D’une part la résistance qui permet à la cellule leucémique d’échapper à la pression thérapeutique des ITK et d’autre part la résistance « intrinsèque » de la cellule souche leucémique par des mécanismes certainement multiples. Ce second niveau de résistance est à l’origine de la récurrence de la LMC lors de l’arrêt du traitement.Cette thèse a consisté à déterminer comment pouvait mourir les cellules de LMC en réponse aux ITK pour mettre en évidence les morts induites et les régulations qui existent entre-elles. De plus, cela a permis d’utiliser les morts non-apoptotiques pour contourner les mécanismes de résistance aux ITK.Nous avons montré pour la première fois en utilisant différents modèles cellulaires de LMC (cellules K562, Lama-84 et AR-230), que l’imatinib (ainsi que les autres ITK nilotinib et dasatinib) induit de la sénescence en plus d’une réponse apoptotique. En absence d’apoptose, par inhibition de cette dernière, la réponse sénescente devient une réponse majeure des cellules de LMC suggérant que l’apoptose a un rôle de « frein » sur la sénescence. L’autophagie activée par les ITK régule négativement la réponse apoptotique alors qu’elle est nécessaire pour une réponse sénescente majeure. Nous avons pu mettre en évidence deux types de sénescences induites par l’imatinib : une sénescence dépendante et une indépendante de l’autophagie. L’autophagie semble donc au cœur de la régulation des morts cellulaires. Puisque les cellules de LMC peuvent mourir par des morts non-apoptotiques, nous avons cherché à éliminer les cellules résistantes par des morts non-apoptotiques. Pour cela différentes molécules ont été utilisées telles que l’acide mycophénolique (MPA), un immunosuppresseur déjà utilisé en clinique. Le MPA en inhibant la synthèse de GTP permet d’induire des dommages à l’ADN et une réponse apoptotique et/ou sénescente. Dans ce contexte, l’autophagie protège la cellule de la réponse apoptotique mais ne protège pas la cellule de la sénescence. Le MPA est au contraire un puissant inducteur d’apoptose sur les cellules primaires. En effet, il induit une apoptose massive des cellules primaires résistantes aux ITK quelque soit le mécanisme impliqué (surexpression de tyrosine kinase, mutation de Bcr-Abl). Le MPA est l’exemple parfait des molécules qu’il nous faut rechercher pour éliminer les cellules résistantes de LMC notamment dans le cas où les patients sont en crise blastique et donc résistants aux thérapeutiques.Ces résultats suggèrent que la sénescence est une des morts qui peut être induite pour dépasser la résistance des cellules cancéreusesChronic Myeloid Leukemia is a myeloproliferative syndrome connected to the acquisition of a chromosomal abnormality t(9;22) leading to the expression of a fusion protein p210 Bcr-Abl of whom the tyrosine kinase activity deregulated is necessary and sufficient to engender the disease.This pathology benefits since 2002 of a therapeutic advance: the tyrosine kinase inhibitors (TKI). This targeted therapeutics, from which imatinib is the front-line, is very effective because 80 % of patients enters in remission. However, 20 % of the treated patients develop primary or secondary resistances which can be dependent or not to the Bcr-Abl oncogene among which some have been characterized. Indeed, CML is now a model to study both oncogenic and resistances mechanisms.Resistance to TKI in CML can be considered on two sides. On one hand the resistance allowing the leukemic cell to escape the therapeutic pressure of TKI and on a second hand the “intrinsic” resistance of Leukemic stem cells by multiple mechanisms. This second level of resistance is at the origin of the CML recurrence.This thesis consisted in determining how could die the CML cells in response to TKI to bring to light cell deaths induced and the regulations existing between them. Furthermore, it allowed exploring the use of non-apoptotic cell deaths to overcome resistance to TKI.We showed for the first time by using CML cell lines (K562, Lama-84 and AR-230), that imatinib (as well as nilotinib and dasatinib) induced senescence besides an apoptotic response. In absence of apoptosis, by its inhibition, senescence becomes a major response of CML cells suggesting that apoptosis is limiting senescence. Autophagy activated by TKI negatively regulates apoptosis while it is necessary for a major senescent response. We were able to bring to light two types of senescence in response to TKI : a senescence dependent and a senescence independent of autophagy suggesting it plays a critical role in cell death regulation.Because CML cells can die by non-apoptotic cell deaths, we used them to eliminate TKI resistant cells. Mycophenolic acid (MPA), an immonusuppressor already used in therapeutic as an immunosuppressive agent has been extensively used. MPA by inhibiting the synthesis of GTP induces DNA damage and apoptotic and\or senescent response. In this context, autophagy protects the cells from apoptotic response but do not from senescence. Conversely, MPA is a powerful inductor of apoptosis on hematopoietic primary cells. Indeed, it induces apoptosis of TKI resistant primary cells whatever the mechanism involved (overexpression of tyrosine kinases or mutation of Bcr-Abl). MPA illustrates the need to look for new molecules to eliminate TKI resistant CML cells, particularly when patients are in the evolved blastic phase of the disease.These results suggest that senescence is one of the deaths which can be used to overcome resistance of cancer cells
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Gallagher, Laura. "Nutrient-dependent effects of the autophagy-lysosomal inhibitor, Chloroquine, on cell death and lysosomal functionality." Thesis, University of Strathclyde, 2017. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=27858.
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There is huge potential for targeting pro-survival autophagy as a cancer therapeutic strategy, but this approach has not yet been fully realised due to the complex involvement of autophagy for cancer biology. In early stages of transformation, autophagy acts as a tumour suppressor, whilst in a developed tumour, autophagy aids cancer cell survival contributing to resistance. This thesis aimed to interrogate the role of autophagy for survival in metastatic breast cancer using the 4T1 mouse mammary carcinoma model. Investigations utilised the autophagy-lysosomal inhibitor Chloroquine (CQ) since this compound is currently in clinical trials for a variety of blood and solid cancers, including breast cancer. Here, the mechanisms of CQ and furthermore, the potential of combining this drug with metabolic targeting strategies were investigated. In doing this, an unexpected resistance mechanism linking glucose metabolism and CQ was uncovered. In clonogenic assays, CQ induced cell death that cooperated with other therapeutic stressors such as ionising irradiation and PI3K-Akt inhibition. CQ and the metabolic stress of serum starvation produced cell death within 24hrs; however, unexpectedly, further glucose starvation or hexokinase inhibition fully rescued cell viability. The cytotoxic effects of CQ were found to be autophagy-independent as knockdown of ATG proteins did not mimic CQ. As the form of cell death in our model did not resemble classical caspase-dependent apoptosis or necrosis, it was hypothesised the cytotoxic effects of CQ were potentially triggering lysosome membrane permeabilisation. Indeed, CQ treatment did lead to marked lysosomal stress and enlargement, suggestive of LMP. In contrast, while CQ was still able to enter and deacidify the lysosome in glucose starved cells, it failed to induce enlargement. Our data indicate that glucose metabolic rate has a profound influence on the efficacy of CQ to target lysosomes and to induce LMP-mediated death. These effects may be reducing clinical outcomes of CQ in cancer cells with reduced glucose metabolism.
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Singh, Subir. "Role of cytochrome P450 in breast carcinogenesis." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/role-of-cytochrome-p450-in-breast-carcinogenesis(ed2c5c1b-d2e9-458b-acc5-3c320cf22ee6).html.
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Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.
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Watanabe, Motonobu. "Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation." Kyoto University, 2009. http://hdl.handle.net/2433/124304.
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Hannigan, Adrienne Michelle. "Investigating the role of the inhibitor of apoptosis protein, Apollon, in the regulation of autophagy in breast cancer cells." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/26673.
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Macroautophagy (autophagy) is a lysosomal process for degrading cytoplasmic proteins and organelles for maintenance of homeostasis as well as for bioenergetic and biosynthetic needs. During nutrient deprivation and chemotherapy, both tumour-related stresses, autophagy is upregulated. The molecules and pathways involved in the
regulation of autophagy in response to these stresses are still not well understood.
Several recent studies have uncovered links between components of autophagy and apoptosis. As there is increasing evidence indicating that many anti-cancer therapeutics affect both autophagy and apoptosis, it is critical to identify the relationships between
these pathways to develop more rational therapies. A previous study identified several Drosophila melanogaster genes with
autophagy-regulating functions. One of these genes was dBruce, a member of the inhibitor of apoptosis (IAP) gene family, which was found to negatively regulate
autophagy in Drosophila cells in vitro and in vivo. The mammalian homologue of dBruce, Apollon, is overexpressed in several cancers, and Apollon knockdown has been
shown to sensitize some cancer cells to chemotherapy. These findings led to the suggestion that Apollon may be a promising target for cancer therapy. As autophagy has been shown to play a role in cancer development and treatment, a link between Apollon
and autophagy may have clinical implications. My hypothesis in this study was that Apollon, the human homologue of dBruce, is
a negative regulator of autophagy in human breast cancer cells. I tested this hypothesis
using three different breast cancer cell lines, SKBR3, BT474 and MCF-7, to determine
whether Apollon knockdown had an effect on autophagy under fed or starved conditions.
After Apollon knockdown, MCF-7 and SKBR3 cells showed a significant increase in GFP-LC3 and/or MDC puncta under both fed and starved conditions. Further analysis in
MCF-7 cells confirmed that Apollon knockdown led to an induction of the complete autophagy process as determined by two autophagy flux assays. These results show that
reducing Apollon expression induces autophagy and thus Apollon is a negative regulator
of autophagy in human breast cancer cell lines. Further studies will be required to determine whether Apollon knockdown-induced autophagy would be beneficial for
cancer therapy.
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Eimer, Sandrine. "Etude des réponses induites par l’erlotinib dans des cellules de lignées de glioblastome." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21822/document.
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Le glioblastome (GBM), tumeur de plus haut grade du système nerveux central (OMS grade 4) a un pronostic très sombre, quelque soit le traitement, lié à une résistance à l’apoptose. L’erlotinib (Tarceva®, OSI 774) est un inhibiteur de la tyrosine kinase du récepteur au facteur de croissance épithélial (EGFR). L’hyper-expression et l’amplification du gène de l’EGFR dans 40 à 60% des GBM, fourni un rationnel pour utiliser l’erlotinib. Nous avons montré sur U87-MG et DBTRG-05MG, deux lignées de GBM, l’absence d’apoptose avec l’erlotinib, liée soit à un déficit en pro-caspase 3, soit à une accumulation d’αB-crystalline bloquant l’activation de la caspase-3. L’absence d’apoptose dévie alors la cellule vers l’autophagie. L’inhibition de l’autophagie par ARN interférents ou par la chloroquine permet d’obtenir une synergie avec l’erlotinib en induisant la mort des cellules tumorales à des doses acceptables.Les GBM ont composition cellulaire hétérogène, avec un petit nombre d’éléments appelés cellules souches cancéreuses (CSC). Douées d’auto-renouvellement, elles participent à la propagation tumorale et à la résistance aux traitements. Nous avons testé l’erlotinib sur trois lignées issues de GBM humains, ayant deux modes de croissance distincts selon les conditions de milieu: en neurosphères (NS) et de type adhérent. Erlotinib a un effet inhibiteur minime sur les trois lignées adhérentes, alors que l’effet est significatif sur les lignées NS, traduisant l’importance de la voie d’EGFR pour les NS. Dans les lignées en NS, l’erlotinib est efficace sur les cellules progénitrices, mais n’a pas d’action ni sur les cellules initiatrices de NS, ni sur les cellules différenciées. L’auto-renouvellement des NS n’est pas non plus altéré. L’association cyclopamine, inhibiteur pharmacologique de la voie de Hedgehog, -erlotinib est synergique en bloquant la croissance et l’initiation des NS, laissant présager une efficacité sur les CSC. Les résultats obtenus sur ces différents modèles permettent d’une part de préciser certains mécanismes de résistance des cellules de GBM, et aussi d’orienter les indications et le choix des traitements susceptibles d’être les plus efficacesGlioblastoma (GBM) is the most common primary central nervous system tumor in adults and the prognosis remains dismal, any treatment used. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in GBM, making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. We showed for U87-MG and DBTRG-05MG, two human GBM cell lines, that erlotinib can’t trigger apoptosis, related either to accumulation of αB-crystallin capable to impair caspase 3 cleavage, or to constitutive deficit for procaspase 3 in DBTRG-05MG. Apoptosis deficit switches the cell to autophagic process. Inhibition of autophagy with RNA interference or chloroquine resulted in sensitization of U87 and allowed a synergistic effect with erlotinib at therapeutic doses.Moreover, GBM showed a heterogeneous cell composition with cancer stem cells, progenitors and more differentiated cells. In this study, we test erlotinib in vitro on other GBM models: three cell lines established from surgically resected GBM specimens, grown along two features adherent and neurospheres. On the three differentiated adhering cell lines, erlotinib had only a moderate activity. Conversely, on neurosphere forming cell lines, erlotinib induced a strong inhibition of cell growth related to the EGFR amplification and EGFR expression. A short erlotinib exposure induced cell death primarily in nestin-positive cells; however it was found without effect on neurosphere initiating activity and self renewal. These results suggest that EGFR pathway activation is essential for the proliferation of GBM progenitor cells but dispensable for stem-like cancer cells self–renewal. As Hedgehog pathway is known to be activated in neural stem cells, we assayed the Hedgehog pathway inhibitor cyclopamine in association with erlotinib. While each drug separately was without effect on sphere initiation, their combination led to a 25 fold decrease in the sphere number (p=0.0004).These in vitro models are convenient to investigate resistance mechanisms in GBM. Furthermore, they focus on the necessity to exploit drug combinations for greatest efficiency
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Xie, Wei. "Transcription Inhibitor Lurbinectedin and Oncolytic Peptide LTX-401 trigger Immunogenic Cell Death and Synergize With Immune Checkpoint Blockade Lurbinectedin Synergizes With Immune Checkpoint Blockade To Generate Anticancer Immunity Tumor Lysis With LTX-401 Creates Anticancer Immunity Autophagy Induction by Thiostrepton Improves the Efficacy of Immunogenic Chemotherapy Oncolysis With DTT-205 and DTT-304 Generates Immunological Memory in Cured Animals." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL072.
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Le cancer est la deuxième cause de décès dans le monde. Malgré l'existence des traitements standards, le développement et la recherche de stratégies thérapeutiques innovantes et de médicaments est toujours nécessaire. La combinaison des médicaments induisant la mort cellulaire immunogène (ICD) et l'inhibition des points de contrôle immunitaire (ICB), semble être un protocole prometteur. Dans cette thèse, nous avons démontré que la Lurbinectedine, un inhibiteur de la transcription nouvellement approuvé pour le traitement du cancer du poumon récidivant, déclenche les caractéristiques de l'ICD dans quatre différentes lignées cellulaires humaines et murines in vitro. Vaccinée par des cellules de fibrosarcome traitées par la lurbinectedine, les souris immunocompétentes sont protégées du rechallenge des tumeurs syngéniques. La lurbinectedine limite la croissance du fibrosarcome transplanté d'une manière immunodépendante. Dans les souris, le fibrosarcome murin (MCA205) transplanté et le cancer du sein, induit par des hormones en combinaison avec des cancérigènes, ont été sensibilisés par la lurbinectedine aux deux ICB : PD-1 et CTLA-4. Il convient de noter que la mémoire immunologique à long terme a été générée chez des souris guéries. En outre, nous avons évalué la capacité anticancéreuse de LTX-401, un peptide oncolytique conçu pour l'immunothérapie locale. Les injections intratumorales séquentielles de LTX-401 retardent considérablement la croissance des tumeurs sous-cutanées MCA205 et TC-1 chez un hôte immunocompétent, mais montrent un effet thérapeutique limité sur les tumeurs syngéniques abscopales. Une seule administration de LTX-401 augmente l'efficacité de l'ICB anti-CTLA-4 ou anti-PD-1 + anti-CTLA-4. De plus, le traitement séquentiel avec LTX-401 et les deux ICB présente une immunité antitumorale systémique à la fois contre la tumeur traitée et la tumeur abscopale. En conclusion, la lurbinectedine et le LTX-401 induisent la mort cellulaire immunogène des cellules cancéreuses et renforcent les effets anticancéreux des inhibiteurs de points de contrôle immunitaires. Ces résultats jettent les bases expérimentales de traitements combinés et peuvent faciliter les conceptions d'essais cliniquesCancer is the second leading cause of death worldwide, despite the existence of standard treatment, innovative therapeutic strategies and drugs are still in urgent demand. The combination of immunogenic cell death (ICD) inducing drugs and immune checkpoint blockade (ICB) seems to be a promising modality. In this thesis, we demonstrated Lurbinectedin, a transcription inhibitor newly approved for relapsed lung cancer treatment, triggers hallmarks of ICD in four different human and murine cell lines in vitro. Vaccinated with Lurbinectedin-treated fibrosarcoma cell protects immunocompetent mice from rechallenge with syngeneic tumours. Lurbinectedin restrains transplanted fibrosarcoma growth in an immune dependent manner. Both transplanted MCA205 cancer and hormone/carcinogen induced breast cancer were sensitized by Lurbinectedin to PD-1 and CTLA-4 double ICBs. Of note, long-term immunological memory was generated in cured mice. Further, we evaluated the anticancer capacity of LTX-401, an oncolytic peptide designed for local immunotherapy. Sequential intratumoral injections of LTX-401 dramatically retards subcutaneous MCA205 and TC-1 tumour growth in immunocompetent host, yet shows limited therapeutic effect of anti-CTLA-4 or anti-PD-1/anti-CTLA-4 ICBs. Moreover, sequential LTX-401 treatment with double ICBs exhibits systemic antitumor immunity to both treated and abscopal tumour. In conclusion, lurbinectedin and LTX-401 induce cancer cell immunogenic cell death and enhance the anticancer effects of immune chekcpoint blockade. These results lay the experimental foundation of combination regiments and may facilitate the clinical trial design
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Aubert, Serge. "Effets multiples du glycérol sur le métabolisme de la cellule végétale non chlorophyllienne." Université Joseph Fourier (Grenoble), 1994. http://www.theses.fr/1994GRE10217.
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Nous avons étudié les effets du glycérol sur la croissance et le métabolisme des cellules végétales non chlorophylliennes, en utilisant des cellules isolées d'érable sycomore (Acer Pseudoplatanus, l. ) et d'Echinochloa. Le glycérol pénètre dans les cellules par diffusion. Il est ensuite phosphoryle en SN-glycérol-3-P qui alimente la respiration, via les réactions terminales de la glycolyse. La néoglucogenèse est impossible car le SN-glycérol-3-P est un inhibiteur compétitif de la glucose-6-phosphate isomérase. Ainsi, la synthèse de saccharose, d'amidon et de composes pariétaux s'arrête. De plus le cycle oxydatif des pentoses cesse d'être alimente, ce qui entraine un arrêt de la synthèse des pentoses et de la réduction des nitrates. Lorsque le glycérol est utilise comme seule source de carbone, la croissance des cellules est alors stoppée. Pourtant le seul fonctionnement de la respiration mitochondriale suffit à entretenir durant plusieurs semaines la survie des cellules, qui ne présentent aucun signe d'autophagie. Par contre, une carence de glycérol entrainera brutalement le processus autophagique. Par ailleurs, nous avons observe que le glycérol entraine l'accumulation d'oligopolysaccharides de types B-1,3 et de phosphohomoserine (un intermédiaire de la synthèse d'isoleucine, issu de l'aspartate) lorsque le milieu de culture contient du saccharose. En utilisant l'homosérine (dont nous avons caractérise le transport actif dans les cellules et la métabolisation) et les herbicides inhibant l'acétolactate synthase, nous avons montre que le site principal de régulation de la synthèse in vivo de l'isoleucine est situe au niveau de la thréonine déshydratase. Une observation a été mentionnée en annexe : l'induction de la respiration insensible au cyanure par les inhibiteurs de l'acétolactate synthase
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Dias, Pedro Gonçalo Guedes. "Modulation of neuronal mitochondrial dynamics, autophagy and huntingtin proteostasis by HDAC inhibitors: Insights for Huntington's disease." Doctoral thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/78858.
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Dias, Pedro Gonçalo Guedes. "Modulation of neuronal mitochondrial dynamics, autophagy and huntingtin proteostasis by HDAC inhibitors: Insights for Huntington's disease." Tese, 2015. https://repositorio-aberto.up.pt/handle/10216/78858.
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Herbert, James Taylor. "The Role of Autophagy and Translation Initiation Factors in Overcoming Resistance to mTOR Inhibitors in Prostate Cancer." Diss., 2013. http://hdl.handle.net/10161/7097.
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Castration resistant prostate cancer (CRPC) causes significant morbidity and mortality around the world and improving treatment options for patients with CRPC is a major concern for biomedical research. Because of the importance of activating mutations in the PI3K/AKT/mTOR pathway in prostate cancer, several mTOR inhibitors have been tested for efficacy in CRPC but despite promising preclinical findings, the results of clinical trials have been disappointing. The findings of several groups, including a clinical trial of RAD001 conducted at Duke, suggest that feedback upregulation of PI3K and autophagy may be potential mechanisms for resistance of CRPC to mTOR inhibitor therapy.
The main goal of this dissertation was to explore these mechanisms in vitro and to determine if combinations of PI3K inhibitors and different classes of mTOR inhibitors can overcome resistance to mTOR inhibitor monotherapy. In particular, we used immunoblotting, reverse phase protein microarrays, polysome profile analysis, cell cycle analysis, and several techniques for determining cell survival and proliferation to explore the differences in survival, proliferation, autophagy, and activity of the AKT, translation initiation, and autophagy cell signaling networks between prostate cancer cell lines treated with different combinations of mTOR and PI3K inhibitors. Our findings revealed that the combination of PI3K and mTOR inhibition leads to a synergistic inhibition of prostate cancer cell survival and cytostasis that is correlated decreased translation rates, hypophosphorylation of 4E-BP1, autophagy, and an uncoupling of normal signaling between AKT and mTOR. We were able produce an effect on cell survival similar to treatment with high doses of mTOR/PI3K inhibitor combinations by inhibiting cap-dependent translation using a non-phosphorylatable mutant of 4E-BP1. In contrast, knocking down two major autophagy genes had little to no effect on the survival of prostate cancer cells treated with PI3K/mTOR inhibitors but did protect from cell death caused by the UPR activator tunicamycin.
We conclude that treatment strategies that target PI3K, mTORC1 and mTORC2 simultaneously have the potential to be clinically useful in CRPC, probably due to the increased inhibition of eIF4E activity and cap-dependent translation when compared to monotherapy with allosteric mTORC1 inhibitors. Although autophagic cell death can be induced in prostate cancer cells, the autophagy observed after inhibition of PI3K and mTOR does not appear to contribute to cell death and is not a major resistance mechanism under these conditions. Nevertheless, we did observe different roles for autophagy in the survival of cells exposed to different types of stressors, and further elucidation of autophagy signaling networks may yet provide useful clinical targets.
Dissertation
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Kolárik, Matúš. "Studium působení tyrosinkinasových inhibitorů a jejich metabolitů na buněčné linie nádorů." Master's thesis, 2021. http://www.nusl.cz/ntk/nusl-448743.
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Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...
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Gooskens, Brigite Teixeira Ribeiro Van Den Wildenberg. "Stents, statins and sirolimus: a new approach to prevent restenosis." Master's thesis, 2018. http://hdl.handle.net/10316/81998.
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Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de MedicinaIntrodução: As doenças cardiovasculares continuam a ser a principal causa de morte no mundo, sendo a doença arterial coronária, responsável por 20%, anualmente, na Europa. O tratamento abrange abordagem conservadora, revascularização miocárdica cirúrgica e intervenção coronária percutânea, muito menos invasiva. Os stents farmacológicos implantados pela intervenção coronária percutânea foram um marco que reduziu significativamente a taxa de reestenose do stent e a trombose do stent associada aos primeiros stents metálicos usados; no entanto, os stents farmacológicos ainda apresentam algumas desvantagens. Além da inibição da proliferação das células musculares lisas, a completa reendotelização do vaso lesado após o implante do stent, com endotélio regenerado, mostrando características e funções morfológicas normais, é um objetivo estratégico para o stent. Uma grande desvantagem do uso de stents farmacológicos com compostos como a rapamicina (sirolimus), um inibidor do mTOR, é que não apenas atua nas células musculares lisas vasculares, mas também nas células endoteliais, o que poderá exacerbar a disfunção endotelial provocada pelo stent. Embora a autofagia possa ser ativada diretamente pela inibição da mTOR, ela pode ser ativada indiretamente, através da proteína quinase ativada por 5 ' monofosfato de adenosina (AMPK). Como as estatinas podem ativar a AMPK e melhorar a função endotelial, reduzindo a inflamação, nós colocamos a hipótese que as estatinas possam reduzir os efeitos deletérios dos stents farmacológicos de sirolimus, nas células endoteliais, melhorando a resposta arterial e reduzindo a taxa de reestenose.Métodos: Para abordar esta questão, utilizámos uma abordagem celular, com uma linhagem celular endotelial cardíaca imortalizada de ratos e uma cultura primária de células endoteliais. Estas células foram tratadas com estatinas (sinvastatina) ou rapamicina separadamente ou em combinação. Em seguida, avaliou-se a atividade endotelial por ensaio de tubulação de matrigel, ensaios de migração e fluxo de autofagia/atividade por western blot.Resultados: Os nossos resultados demonstraram que, quando em combinação com a rapamicina (ou sirolimus), a sinvastatina reverte parcialmente os efeitos negativos da rapamicina no potencial angiogénico das células endoteliais, observado por um aumento na capacidade de migração e tubulação. Além disso, observámos que a sinvastatina promoveu a autofagia.Discussão: O aumento da angiogénese pode ser atribuído a um aumento de neovasos de paredes finas e frágeis, que podem servir para o recrutamento de leucócitos para áreas de alto risco da placa. Por outro lado, a capacidade das células endoteliais para a angiogénese correlaciona-se com uma resposta arterial adequada e um endotélio funcional. Observou-se ainda um aumento da capacidade de migração das células endoteliais, o que significa que elas responderam a um estímulo quimiotácito e migraram através de uma barreira física em direção a ele, o que é desejável quando há lesão endotelial, nomeadamente aquela induzida pela impantação de um stent.Os resultados sugerem que comparando com a rapamicina, a adição de sinvastatina promove a autofagia, que tem sido associada à angiogénese nas células endoteliais, com melhor resposta arterial. Conclusão: Estes resultados abrirão caminho para novos entendimentos sobre os efeitos das estatinas nas células vasculares, e potencialmente um stent com eluição de estatina, o padrão na prática clínica, para um menor risco de reestenose e disfunção endotelial.
Introduction: Cardiovascular diseases remain the leading cause of death worldwide, being coronary artery disease accountable for up to 20% annually in Europe. The treatment encompasses conservative management, coronary artery bypass graft and the much less invasive percutaneous coronary intervention. The drug-eluting stents deployed by percutaneous coronary intervention were a milestone that significantly reduced the stent restenosis rate and stent thrombosis associated to the first-ever used bare-metal stents; however, drug-eluting stents still present some drawbacks. Besides smooth-muscle cells proliferation inhibition, achieving complete reendothelialization of injured vessel after stenting, with a regenerated endothelium showing normal morphologic characteristics and functions, is a strategic objective for a stent. One major disadvantage of the use of drug-eluting stents with compounds such as rapamycin, also known as sirolimus, a mTOR inhibitor, is that not only acts on the vascular smooth muscle cells, but also on endothelial cells, which might exacerbate the endothelial function impairment when the stent is deployed. Although autophagy can be directly activated by mTOR inhibition, it can be activated indirectly, namely through 5’ adenosine monophosphate-activated protein kinase (AMPK). Since statins can activate AMPK and improve endothelial function, reducing inflammation, we hypothesize that statins can reduce the detrimental effects of sirolimus-eluting-stents on endothelial cells promoting a beneficial increase in endothelial function and consequentially a better arterial healing response, and reduced stent restenosis.Methods: To address this question, we used a cell-based approach, with an immortalized mouse cardiac endothelial cell line and an endothelial cell primary culture. These cells were treated with statins (simvastatin) or rapamycin separately or in combination. Afterwards we evaluated endothelial activity by matrigel tubulation assay, migration assays and autophagy flux/activity by western blot. Results: Our results demonstrated that when in combination with rapamycin (or sirolimus), simvastatin partially reverts the negative effects of rapamycin in the endothelial cells angiogenic potential, observed by an increased tubulation and migration capacity. Moreover, we observed that simvastatin potentiates autophagy regulation.Discussion: The increase on angiogenesis might be attributable to an increase of thin-walled and fragile neovessels that may serve as a pathway for recruitment of leukocytes to high-risk areas of the plaque. On the other hand, the capacity of endothelial cells for angiogenesis correlates with a proper arterial healing response and functional endothelium. There was an increase of the ability of endothelial cells to migrate and subsequently close a wound, what means that they were able to follow chemo-attractant and migrate through a physical barrier toward it, which is a desirable quality when there is endothelial damage, such as when a stent is deployed.Comparing to rapamycin alone, the addition of simvastatin seems to further increase autophagy, that has been associated with angiogenesis in endothelial cells, and with a better arterial healing response. Conclusion: These results will pave the way for new understandings on the effects of statins on the vascular cells, and potentially a statin-eluting stent the default one on clinical practice, for a lower risk of restenosis and endothelial dysfunction.
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Yu-WeiChiu and 邱宥維. "B1 as an autophagy inhibitor effect on RGNNV replication and host AMPK autophagy signaling pathway." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/rb4432.
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Chen, Man-Chin, and 陳曼菁. "Connexin 43 inhibits tumor growth by inducing autophagy." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/9ydu8z.
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碩士中國醫藥大學
基礎醫學研究所碩士班
102
Autophagy is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol. Tumor cells frequently display lower levels of basal autophagic activity than their normal counterparts and fail to increase autophagic activity in response to stresses. Directly cell-to-cell communication is critical for maintaining homeostasis. Thus, the regulation of connexin protein levels is important. Gap junction channels are composed of connexons (or hemichannels) which are comprised of integral four-pass trans-membrane protein called connexins. Connexin 43 (Cx43) is the most extensively expressed in many cell types. The degradation mechanisms for connexins have been demonstrated in autophagosomal degradation pathway. The presence of functional gap junctions is highly relevant for autophagy. Here, we decreased Cx43 expression by short-hairpin RNA technology and examined their activities in murine cancer cells with serum starvation status. The autophagic markers were decreased after tumor cell downregulated Cx43. The loss of Cx43 expression decreased autophagy, which displays lower levels of basal autophagic activity in a majority of cancers. To study the pathway underlying Cx43-induced effects, we found that Cx43 induced a significant increase in mitogen-activated protein kinases (MAPK) signal pathways which involved in autophagy. Herein, our findings that Cx43 in controlling tumor growth may induce autophagic signal pathways.
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Lai, Wei-Ting, and 賴瑋婷. "Connective Tissue Growth Factor inhibits Cancer Metabolism and Autophagy." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/5hpr97.
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博士國立臺灣大學
臨床牙醫學研究所
105
In Taiwan, oral cancer (in particular oral squamous cell carcinoma, OSCC) is the fourth leading cause of death in males, and is one of the causes with high death rate worldwide. In this study, we seek to uncover the underlying mechanism of OSCC and help the development of new therapeutic strategies for OSCC. First, we analyzed 82 OSCC cases and confirmed that OSCC patients with high ataxia telangiectasia mutated interactor (ATMIN) expression showed poor differentiation, higher TNM stages, greater lymph-node metastasis, and shorter accumulated survival time. Evidence from a buccal orthotopic implantation mouse model showed that silencing of ATMIN expression reduced lymph node metastasis and prolongs the survival of mice. ATMIN is now considered as a poor prognosis biomarker, and our study results help to identify possible therapeutic targets of downstream genes for designing effective therapeutic strategies for OSCC. Second, my previous studies showed that connective tissue growth factor (CTGF/CCN2) significantly blocked glycolysis, mitochondrial oxidative phosphorylation (OXPHOS), and adenosine triphosphate (ATP) production. Moreover, CTGF showed no effects in normal bronchial and oral epithelial cells. We demonstrated that CTGF decreased glycolysis, mitochondrial oxidative phosphorylation, ATP generation and mitochondrial DNA (mtDNA) copy number by increasing the degradation of mitochondrial transcription factor A (mtTFA) through ubiquitin proteasome pathway and in turn reduced migration and invasion of OSCC cells. Autophagy can promote cancer cell survival or resistance under conditions of poor nutrient supply, chemotherapy or target therapy. In this study, we demonstrated that CTGF inhibited late stage autophagy through blocking autophagosome-lysosome fusion under starvation condition. We further showed that cotreatment of CTGF could markedly enhance the therapeutic efficiency of Erbitux in vivo Finally, we showed that overexpression of CTGF significantly decreased Rab5A expression, which is essential for completion of autophagy. Furthermore, expression of a wild type or constitutive active form of Rab5A could restore CTGF-inhibited autophagy flux. We investigated the clinical importance of CTGF-Rab5A axis in OSCC patients, the results showed that high Rab5A mRNA expression was significantly associated with an advanced clinical pathological TNM stage. A reverse correlation between CTGF and Rab5A was also demonstrated. The findings in this study demonstrated that ATMIN and Rab5A can be poor prognostic biomarkers in OSCC. I believe that CTGF has the potential to be developed as an additive therapeutic agent for cancer treatment in the future.
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Hsieh, Shang-Ying, and 謝尚穎. "Development of a Novel Quinoline-Based Autophagy Inhibitor for Effective Cancer Treatment." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/58qd94.
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Chiao, Ming-Tsang, and 矯明昌. "The Mechanism of Autophagy Induced by Histone Deacetylase Inhibitor (SAHA) in Glioblastoma Stem Cell." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/64775778137449422385.
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博士中山醫學大學
醫學研究所
101
We report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133+-GSC) and CD133- (CD133--GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with xenografted tumors derived from CD133--GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133+-GSC-derived tumor xenografts. Subsets of CD133+-GSC but not CD133--GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133--GSC and exclusively dispersed within the tumor tissues. Although, the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells. In addition, although Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been used in clinical trials for cancer therapies, its pharmacological effects occur through a poorly understood mechanism. Here, we report that SAHA specifically triggers autophagy and reduces cell viability via promotion of apoptosis in the late phase of glioblastoma stem cells (GSCs). Using a cell line cultured from a glioblastoma biopsy, we investigated the properties and effects of GSCs under SAHA treatment in vitro. In vivo xenograft assays revealed that SAHA effectively caused tumor growth slowdown and the induction of autophagy. SAHA was sufficient to increase formation of intracellular acidic vesicle organelles, recruitment of LC3-II to the autophagosomes, potentiation of BECN1 protein levels, and reduced SQSTM1 levels. We determined that SAHA triggered autophagy through the downregulation of AKT-mTOR signaling, a major suppressive cascade of autophagy. Interestingly, upon depletion or pharmacological inhibition of autophagy, SAHA facilitates apoptosis and results in cell death at the early phase, suggesting that SAHA-induced autophagy functions probably act as a prosurvival mechanism. Furthermore, our results also indicated that the inhibition of SAHA-induced autophagy using chloroquine has synergistic effects that further increase apoptosis. Moreover, we found that a reduced dose of SAHA functioned as a potent modulator of differentiation and senescence. Taken together, our results provide a new perspective on the treatment of GSCs, indicating that SAHA is a promising agent for targeting GSCs through the induction of autophagy.
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Chang, Ya-Ping, and 張雅評. "Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and inducing autophagy." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/93245095721695579026.
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博士國立宜蘭大學
生物技術與動物科學系
103
The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1β and plays important roles in the development of inflammation-related disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy.
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Lin, Pei-Jung, and 林珮蓉. "Arsenic treatment inhibits cell autophagy and leads to the occurrence of apoptosis." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/18121488819692751862.
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碩士國立清華大學
分子與細胞生物研究所
104
Arsenic is one of the environmental pollutants and a well-known carcinogen. Previous studies indicated that cytotoxicity caused by arsenic is recognized through generating oxidative stress, attenuating mitochondrial membrane potential, or influencing cell cycle to lead apoptosis. This study investigates the mechanism involved in the arsenic-induced cell death in Human Embryonic Kidney 293 (HEK 293) cells. Upon arsenic stimulation, PI3K/Akt signaling pathway is inhibited and leads to cytochrome c release from mitochondria which activates caspase 3 and apoptosis. However, PTEN is not participated in the PI3K/Akt signaling pathway. PTEN expression is reduced by arsenic treatment. Noticeably, the loss of PTEN is not due to the degradation of the protein, but presents in the insoluble fraction of the cells. The factor that assists protein refolding, heat shock protein 70 (Hsp 70), was also found in insoluble fraction. Concurrently, arsenic treatment inhibited the occurrence of autophagy in HEK 293 cells and caused an accumulation of LC3 in the cells. Administration of lysosomal activator (rapamycin) reduces the PTEN level in either soluble or insoluble fraction of the cells. The blockade of autophagy via reducing lysosomal activity caused the reduction of PI3K/Akt activity and led cells to apoptosis. Addition of rapamycin activates lysosomal activity and allows the cells to go through autophagy pathway. Our results reveal that arsenic treatment inhibits the lysosomal activity, and thus blocks the autophagy pathway. This blockade further reduces the activity of PI3K/Akt signaling pathway and leads to mitochondria-mediated apoptosis.
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MacCallum, S., M. J. Groves, J. James, K. Murray, V. Appleyard, A. R. Prescott, Abed Alnaser A. A. Drbal, et al. "Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6." 2013. http://hdl.handle.net/10454/7250.
Full textAbstract:
NoTenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.
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Liao, Ji-Der, and 廖記德. "Reversine inhibits cell growth and induces apoptosis and autophagy in lung cancer cells." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/34764319223200168724.
Full textAbstract:
碩士中華醫事科技大學
醫學檢驗生物技術系碩士班
101
Reversine ,2-(4-morpholinoanilino)-6-cyclohexylaminopurine, a small synthetic purine analogue, has been used for stimulating stem cell dedifferentiation. It has been reported that reversine is effective in tumor suppression, but it’s effect in lung cancer cells remains unclear. We treated lung cancer cell lines A549, H23 and H1299 with reversine and disseted the related pathways. First, we demonstrated reversine inhibited cell growth and colony formation. Further results revealed reversine induced cell cycle arrested at G2/M phase and formed polyploidy. The expressions of Aurora-A, Aurora-B, p-Akt(Ser473), p-GSK-3alpha(Ser21) and p-GSK-3beta(Ser9) were down-regulated The presence of cleaved caspase-3 and PARP demonstrated reversine in reversine treated cells could induce apoptosis. That the increased LC3-II protein was present after reversine treatment implied that the autophagy is also involved. Taken together, reversine suppressed cell growth of lung cancer cells and induced apoptosis as well as autophagy, which may be an unique advantage for developing novel therapeutic agents for treatment of lung cancers in the future.
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Yi, Wan-Chien, and 萬建億. "Antipsychotic Clozapine Inhibits HCT116 Colorectal Cancer Cell Growth through ERK-Autophagy Signal Pathway." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/80651638753308842423.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
99
Clozapine is an atypical antipsychotic used to treat refractory schizophrenia clinically. Some previous researches as well as clinical statistics indicated that schizophrenia patients taking antipsychotics have less incidence of developing cancer than normal people. Antipsychotics can inhibit cancer cell growth, but the mechanisms still need to further elucidate. Colorectal cancer is the third leading cause of mortality in western countries. The development of colorectal cancer is often associated with gene mutations such as APC, β-catenin and KRAS. In this study, human colorectal cancer cell line HCT116 is used to investigate antitumor ability and molecular mechanisms of clozapine. Preliminary data showed that clozapine inhibited proliferation of HCT116 in both time- and dosage-dependent manners by MTT and clonogenic assay. Clozapine also led to cell cycle arrest at G0/G1 phase and related cyclin-dependent kinase inhibitors p21 and p27 increased while protein expression of CDK6 and pRb (Ser795) decreased. Clozapine did not induce apoptosis by Annexin-V and western blot. However, clozapine induced autophgy (Type II cell death) by increasing LC3II, beclin-1 and Atg5. ERK inhibitor PD98059 inhibited the clozapine-induced increase of LC3II. It is suggested that the effect of clozapine on autophagy is mediated by ERK pathway. It should be further examined whether ERK-autophagy pathway involved in inhibition of clozapine on HCT116 cancer cell growth.
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Chang, Yu-Teng, and 張譽騰. "Flavonoids Participate in the Regulatory Mechanisms of Antizyme Inhibitor in HL-60 Autophagy/ Differentiation/ Activation/ Apoptosis." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/76090874278192234590.
Full textAbstract:
碩士國立中興大學
生命科學系所
104
Antizyme inhibitor (AZI) has been thought to promote cancer cell proliferation, it’s caused between with antizyme (AZ) and ornithine decarboxylase (Ornithine decarboxylase, ODC; EC 4.1.1.17) regulatory mechanism. Flavonoids source from fruits, vegetables, tea, wine, seeds or roots of plants, widely considered to have antioxidant or anti-inflammatory response effect. In our study, we chose four kinds of flavonoids: Epigallocatechin gallate (EGCG)、 Baicalein、 Myricetin、 7,8-Dihydroxyflavone (7,8-DHF), explore its toxic effect in HL-60 immunity cancer cell, and hoped that in the future can be further developed into new drugs for the treatment of leukemia or other cancers. Above these four drugs have been found to inhibit the ornithine decarboxylase enzyme activity, in our laboratory previous study have found catechin composition were impacted AZI protein structure, so we transfected AZI gene into HL-60, then compare whether those flavonoids affect AZI structure and function further impact toxic effect in cells. Moreover, many researches revealed TPA besides caused HL-60 differentiate to macrophage and induced autophagy, activation, differentiation and apoptosis. Based on these study we treat both EGCG and TPA in HL-60 and compare with empty vector and overexpression of AZI. Our data demonstrate those four drugs both could cause HL-60 apoptosis, but EGCG and myricetin caused overexpression of AZI more cell death than empty vector. Co-treated with TPA and EGCG compare with TPA only increase autophagy, activation, differentiation and apoptosis, and overexpression of AZI more strengthened on autophagy, activation, differentiation and apoptosis in HL-60. These results proved that TPA can cause HL-60 differentiation, and after add EGCG accelerates differentiation to cell death ahead of time. Therefore, in the future treatment of human leukemia, by TPA can mix with EGCG as an adjunct to anti-cancer therapy as a guideline.
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Wu, Mei-Yi, and 吳玫憶. "Chloroquine, An Autophagy Inhibitor, Enhances The Cytotoxicity of Gefitinib in Non Small Cell Lung Cancer Cells." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/69956317540921811835.
Full textAbstract:
碩士國立陽明大學
藥理學研究所
100
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, are effective for patients with non small cell lung cancers (NSCLCs). However, patients eventually develop drug resistance. Autophagy, one of the processes to degrading cellular proteins or organelles, reportedly facilitates anti-cancer treatments, including radiation and chemotherapies. In my thesis, two aims were focused, one was the involvement of autophagy in gefitinib-resistance and the other was the effect of chloroquine (CQ), an autophagy inhibitor by preventing formation of autophagolysosomes, on gefitinib-resistance. To serve this purpose, a novel acquired gefitinib-resistant cell line (PC-9/gef) was developed by continuous exposure of the parental PC-9/wt NSCLC cells (containing EGFR exon 19 deletion) to escalating concentrations of gefitinib. Three resistant clones of PC-9/gef cells were identified, including PC-9/gef B4, E3 and E7 cells. Using SRB assay, PC-9/gef cells were more resistant to gefitinib than PC-9/wt cells. The IC50 of gefitinib of PC-9/gef cells was 150-fold more than that of PC-9/wt cells. Compared with PC-9/wt cells, PC-9/gef B4 cells had higher basal LC3-II levels. 3-Methyladenine (3-MA, an early-phase autophagy inhibitor) ameliorated the LC3-II levels in both cells. Compared with PC-9/wt cells, PC-9/gef B4 cells appeared to be more sensitive to CQ-induced elevation in LC3-II levels. These data indicate that PC-9/gef B4 cells had a higher level of autophagy. At the same time, 3-MA inhibited cell survival of both cells in a concentration-dependent manner. However, CQ induced a more significant cytotoxicity in PC-9/gef B4 cells compared with PC-9/wt cells. These data suggest that autophagy may play a pro-survival role in cancer cell survival; the survival of PC-9/gef B4 cells appears to be more dependent on autophagy. In contrast to the 150-fold difference in IC50 of gefitinib, gefitinib concentration-dependently increased LC3-II levels in both cells; however, LC3-II 8 elevation by low dose of gefitinib were more evident in PC-9/gef B4 cells. The data from immunostaining supported this notion that more LC3-II puncta were observed in PC-9/gef B4 cells than in PC-9/wt cells. Furthermore, CQ enhanced gefitinib-induced LC3-II levels in both cells. Gefitinib potently reduced the cell survival of PC-9/wt cells; CQ was unable to potentiate gefitinib-induced cytotoxicity. As to PC-9/gef B4 cells, gefitinib alone induced moderate cell death of PC-9/gef B4 cells; CQ sensitized PC-9/gef B4 cells to the gefitinib-induced cytotoxicity. The cytotoxic mechanisms were investigated by measuring caspase-3 activation. Gefitinib induced significant activation of caspase-3 and CQ did not potentiate gefitinib-induced apoptosis in PC9/wt cells. In contrast, in PC-9/gef B4 cells, gefitinib induced slight increases in active caspase-3 levels ; CQ plus gefitinib showed significant elevation in caspase-3 levels, indicating that CQ may reverse resistance in PC-9/gef B4 cells. An in vivo model xenografting was performed using implanting PC-9/wt cells and PC-9/gef B4 cells. Oral administration of gefitinib significantly reduced the tumor growth of PC-9/wt cells. Combination of gefitinib plus CQ did not further suppress the tumor growth. In contrast, oral administration of gefitinib slightly reduced the tumor growth of PC-9/gef B4 cells. Combination of gefitinib plus CQ further suppressed the tumor growth. The pharmacodynamic study showed augmentation of caspase-9 activation in PC-9/gef B4 tumor tissues in mice treated with gefitinib and CQ. In conclusion, my study showed that PC-9/gef cells have higher basal level of autophagy, which may play a pro-survival role. Furthermore, CQ potentiated gefitinib-induced cytotoxicity in PC-9/gef B4 cells, indicating that CQ may partially reverse the gefitinib resistance. Accordingly, in addition to the consistent activation of ERK pathway in gefitinib resistance which can be overcome by ERK inhibitors, such as AZD6244, CQ and autophagy inhibitors may be a therapeutic strategy for gefitinib resistance.
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Yu, Chen-Lin, and 尤振霖. "Pterostilbene Inhibits the Proliferation of HCC Cells in vitro by Inducing Autophagy and Apoptosis." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/n6d8jb.
Full textAbstract:
碩士中山醫學大學
醫學檢驗暨生物技術學系碩士班
102
Petrostilbene is a natural dimethyl analog of resveratrol and found in many grapes and berries, with multiple pharmacologic activities, including anti-inflammation, anti-oxidation and cancer prevention. Many studies showed that pterostilbene can induce apoptosis and autophagy in various cancer cell lines and further inhibit their viability. However, the exact mechanism of pterostilbene-aused growth inhibition in hepato- cellular carcinoma (HCC) cell lines remains unclear. The main goal of this study is to elucidate how pterostilbene inhibit cell proliferation of HCC cells. Results from MTT assay and DAPI staining showed that pterostilbene caused both dose- and time-dependent inhibition of viability of both cells while the morphology of both cells was changed into a more apoptotic fashion. Cell cycle analysis showed that the phase distribution was changed by pterostilbene treatment. However, the expression of caspase 3, 8 and 9 were not affected, indicating that caspase-independent apoptosis may be activated. Meanwhile, results form AO staining showed that pterostilbene treatment can increase the percentage of AVOs positive cells in both dose- and time-dependent manner. Furthermore, the expression level of LC3-II was also increased in both dose- and time-dependent manner by pterostilbene. In summary, pterostilbene inhibit the cellular viabilities of Huh 7 and SK-Hep 1 via cell cycle arrest and the activation of autophagy.
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Cheng, Hui-Wen, and 鄭惠文. "Thioridazine, an antipsychotic agent, inhibits cancer stem cell growth and induces autophagy of Glioblastoma Multiforme." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/03732371190588805985.
Full textAbstract:
碩士國立陽明大學
生物藥學研究所
102
Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by increased proliferation and resistance to chemotherapy and radiotherapy. Glioblastoma stem cells (GSCs) have been proposed to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Currently, temozolomide is the only FDA-approved treatment for GBM. Therefore, there is an urgent need to discover novel candidate therapeutic drugs for GBM. Here, we identified thioridazine, an anti-psychotic drug, as a potential treatment for GBM via the Connectivity Map database. Thioridazine can cross the blood-brain barrier (BBB), providing a significant advantage over current drug delivery limitations. We demonstrate that thioridazine inhibits the cell viability of both GBM cells and GSCs. Thioridazine induces autophagy in GBM cells and GSCs, as evident by the up-regulation of LC3II, and up-regulates AMPK activity. Moreover, thioridazine induces ER stress in GBM cells. Finally, thioridazine suppresses GBM tumorigenesis and induces autophagy in vivo. The data suggest that thioridazine-induced autophagy has an anti-cancer effect in GBM through activation of AMPK. In summary, we repurpose the anti-psychotic drug, thioridazine, as a potent anti-GBM and anti-GBM cancer stem cell agent for potential clinical trial in the future.
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Schafranek, Lisa Rhiannon. "Assessment of critical survival mechanisms exploited by BCR-ABL1+ cells to evade tyrosine kinase inhibitor-induced death: determination of novel therapeutic targets in chronic myeloid leukaemia." Thesis, 2014. http://hdl.handle.net/2440/92213.
Full textAbstract:
Chronic myeloid leukaemia (CML) is a clonal myeloid proliferative disease that results from constitutive activation of the Bcr-Abl oncoprotein, which disrupts normal cellular signalling potentiating the survival and maintenance of BCR-ABL1+ cells. Tyrosine kinase inhibitors (TKIs), like imatinib, have revolutionised the treatment of CML and have become the model for therapy in other cancers. Imatinib treatment also founded the paradigm that potent and continuous dosing is required for optimal patient response in patients with CML. In contrast to imatinib, the second generation TKI dasatinib has a short half-life of only 3-5 h, nevertheless a once daily dosing regime is sufficient to achieve equivalent responses to twice daily dosing suggesting that continuous and complete inhibition of Bcr-Abl may not be required for optimal response to TKI therapy. Despite initial studies indicating that a very brief exposure to a potent dose of TKI is sufficient to induce cell death in BCR-ABL1+ cells, recent studies have attributed this to sustained low-level inhibition of Bcr-Abl signalling due to inadequate drug washout. As reported in this thesis, experiments with low dose dasatinib treatment, which does not completely inhibit Bcr-Abl phosphorylation but is sufficient to induce cell death, demonstrated inactivation of STAT5 as a sensitive measure of Bcr-Abl activity. Here, it was also confirmed that <1 h exposure to potent TKI with adequate drug washout is insufficient to commit BCR-ABL1+ cells to death and it is established for the first time that at least 2 h of Bcr-Abl kinase inhibition are required. Furthermore, combinations of efficient TKI washout with specific inhibitors of STAT5, JAK and ERK ascertained sustained inhibition of pSTAT5, potentially independent of JAK2, as the determinant of commitment to cell death. Together, this research established that continuous, complete inhibition of Bcr-Abl is not required to induce cell death, but that continuous blockade of STAT5, indicative of low-level threshold Bcr-Abl inhibition, is essential, thus challenging the imatinib paradigm. Although most CML patients respond well to imatinib, only 40% of patients achieve a complete molecular response and some patients develop resistance. Blockade of Bcr-Abl signalling can drive cells to develop new survival mechanism, and amongst others, autophagy and the acquisition of extrinsic survival signalling have been implicated in resistance to therapy and/or persistent disease. Studies presented in this thesis define a role for the activation of autophagy in response to tyrosine kinase inhibition of Bcr-Abl. Induction of autophagy by TKI was confirmed using established markers of autophagy, such as the conversion of LC3-I to LC3-II, degradation of p62 and cellular morphology. Blockade of anti-apoptotic proteins Bcl-2 and Bcl-xL along with activation of stress response pathways were revealed as potential mechanisms of autophagy induction, however, further investigation into these pathways is required. Importantly, the data presented here also established clarithromycin as a novel inhibitor of TKI-induced autophagy, advocating combination treatment with TKI therapy in resistant patients. Recent observations that overexpression of cytokines and their receptors may contribute to BCR-ABL1+ cell persistence in CML patients undergoing TKI therapy. Here, the expression of IL-3 and GM-CSF cytokine receptors in BCR-ABL1+ cell lines and chronic phase CML CD34+ progenitor cells was established and signalling through those was confirmed to maintain STAT5 survival signalling, thereby protecting cells from TKIinduced death. Inhibition of JAK2 with ruxolitinib inhibited cytokine-dependent, but not Bcr-Abl-dependent, activation of STAT5 and neutralised cytokine-induced protection from cell death while having little effect in the absence of cytokines. Together, the findings of this thesis established the critical mechanisms in Bcr-Abldependent and -independent signalling that may also be targeted in combination therapeutic approaches and provides an in-depth understanding of the potential clinical effectiveness of dose reductions during dasatinib therapy. These studies will have broad implications for the ongoing development of therapeutic strategies in CML, particularly in the setting of TKI-resistance, and will aid the goal of achieving a curative treatment for patients with CML.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014
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Chih-YunLiu and 劉知耘. "Targeting survivin by a novel small molecule inhibitor, YM155 induces autophagic cell death in human breast cancer cells." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/24942197536310680653.
Full textAbstract:
碩士國立成功大學
藥理學研究所
101
Despite hormone therapy, targeted therapy and chemotherapy have been developed to target different types of breast cancer; the current breast cancer treatments still have several limitations and undesired side-effects. Thus, it is important to develop novel strategies to treat breast cancer. Survivin (BIRC5) is a member of the inhibitor-of-apoptosis proteins family, and it has been shown to play important role in breast cancer development and progression. YM155 is a novel small molecule inhibitor of survivin. Despite YM155 is currently undergoing different phase II clinical trials including studies in patients with breast cancer, its effectiveness in targeting the estrogen receptor (ER) positive Tamoxifen-resistant breast cancer was seldom determined in the past. In addition, it is still unclear whether YM155 exhibits differential anti-breast cancer functions in different breast cancer subtypes. The purpose of this study is to determine the effectiveness of YM155 in targeting various types of breast cancer and its differential molecular mechanism of action in different breast cancer cell lines. Here, YM155 is equally effective in targeting both the parental ER-positive Tamoxifen-sensitive MCF7 and the MCF7-dervided ER-positive Tamoxifen-resistant breast cancer cells in vitro. YM155 is also effective in targeting the triple-negative MDA-MB-231 breast cancer cells. Surprisingly, Western blot analysis, immunofluorescent microscopy and autophagy/apoptosis inhibition assay revealed that targeting survivin by YM155 induced autophagic cell death, but not caspase-3 dependent apoptosis, in most of the tested breast cancer cell lines; despite it is widely believed that survivin inhibits apoptosis through physical interactions with caspase-3. Interestingly, YM155 also induced autophagy-dependent DNA damage in the treated breast cancer cells. Taken together, targeting survivin by YM155 induces autophagic cell death in breast cancer cells. Importantly, YM155 is a promising anti-cancer compound that has potential for the management of various types of breast cancer.