Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

The unfolded protein response (UPR) is a cellular mechanism whose primary functions are to sense perturbations in the protein-folding capacity of the endoplasmic reticulum and to take corrective steps to restore homeostasis. Although the UPR is conserved across all eukaryotic cells, it is considerably more complex in mammalian cells, due to the presence of three interconnected pathways triggered by separate sensor proteins, a translation attenuation mechanism, and a negative feedback loop. The mechanisms of these interacting biochemical pathways in the mammalian UPR allow for a better fine-tuning of the response than in the case of lower eukaryotes, such as yeasts. The present thesis develops a quantitative mathematical model for the dynamics of the UPR in mammalian cells, which incorporates all the proteins and interactions between them that are known to play a role in this response. This model can be used to provide quantitative information about the levels of its components throughout the response, and to analyze the ramifications of perturbations of the UPR. The model uses a system of ordinary nonlinear differential equations based on biochemical rate equations to describe the dynamics of the UPR as a network of interacting proteins and mRNAs. An early model is presented as a first attempt to investigate the UPR network and construct an inclusive wiring diagram, as well as suggesting a framework to model the differential equations. Then, a refined, quantitative model is designed based on experimental data collected on Mouse Embryonic Fibroblasts treated with Thapsigargin to induce stress and trigger the UPR. The model defines the differential equations and determines the unknown kinetic parameters by optimizing the fit of the system's solution to the experimental data. It includes the UPR's intrinsic feedback loops and allows for the integration of various forms of external stress signals. To the best of our knowledge, it is the first, data-validated, quantitative model in the literature for the UPR in mammalian cells. The last chapters of the thesis address, from a modeling point of view, two important questions for the UPR: (1) cell survival versus apoptosis; and (2) incompleteness of the biological wiring diagram. Recent experimental results show that the UPR is capable of producing qualitatively different results leading to cell survival or death depending on the nature, strength, and persistence of the inducing stress. This thesis proposes several approaches by which the equations can be modified to model the transition from adaptation to apoptosis as a dynamic switch, while taking into account the various hypotheses of cell death mechanisms. Finally, we use recently-developed computational algebra techniques to infer an optimal structure of the UPR network, based solely on the experimental data; the resulting wiring diagram provides insights on elements of the structure of the model that may have been overlooked during the classical (mechanistic) approach to our original data-based model.

alpha-TEA (alpha-tocopherol ether linked acetic acid) has been shown to induce apoptosis in human prostate, ovarian and breast cancer cells in culture and in xenograft models by promoting pro-apoptotic pathways and inhibiting anti-apoptotic pathways. Studies investigated the ability of alpha-TEA to induce apoptosis in human hematological malignant cell lines Jurkat, Raji and U266, representing T cell leukemia, B cell lymphoma and multiple myeloma, respectively. The three cell lines were cultured in the presence of different concentrations of alpha-TEA for different time periods, and examined for apoptosis by annexin V – FITC analyses, DAPI staining, and western blotting for poly (ADP-ribose) polymerase cleavage. alpha-TEA induced apoptosis in all three cell lines in a dose and time dependent manner. Levels of pro-apoptotic molecules DR5, c-Jun N-terminal protein kinase (JNK), C/EBP homologous protein (CHOP), caspase 9, and caspase 3 were upregulated in alpha-TEA treated cells in comparison to vehicle controls. Caspase 8 was activated in Jurkat and U266 cells but not in Raji cells. Apoptosis and pro-death signaling mediators were blocked by ceramide inhibitor, desipramine. The anti-apoptotic nuclear factor kappa B (NF-[kappa]B) signaling pathway was down-regulated in alpha-TEA treated Raji and U266 cells. Combinations of omega-3 fatty acid docosahexaenoic (DHA) and alpha-TEA significantly enhanced apoptosis in Jurkat cells in comparison to single treatments and vehicle control. In summary, alpha-TEA induced apoptosis in the malignant hematological cell lines is via shared and distinct pathways. ASMase/ceramide-mediated JNK activation and endoplasmic reticulum (ER) stress mitochondrial dependent apoptosis are involved in alpha-TEA induced apoptosis in the three cell lines; however, the cell lines exhibit cell type-specific responses to alpha-TEA: activation of death receptor/caspase 8 pathway is involved in Jurkat cells, suppression of NF-[kappa]B signaling is involved in Raji cells, and the U266 cells share both of these pathways for the induction of apoptosis. / text

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Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is utilized by cells to adapt to inter- and intra-cellular changes. There is a burgeoning literature showing that the human endogenous retroviral envelope glycoprotein, Syncyin-1, oxidative stress and reactive oxygen species participate in the pathogenesis of multiple sclerosis (MS). I investigated the contribution of Syncytin-1-induced ER stress in MS and its animal model, experiment autoimmune encephalomyelitis (EAE). The prototypic ER stress biomarker, XBP-1 spliced variant (XBP-1/S), was increased in cerebral white matter of MS patients compared to non-MS controls and was correlated with Syncytin-1 expression. Syncytin-1 over-expression caused glia cytotoxicity but was mitigated by the ROS scavenger, crocin. Treatment with crocin on day 7 post-EAE induction ameliorated EAE disease severity in mice by reducing EAE pathology. Herein, I demonstrate that crocin attenuates Syncytin-1-induced ER stress in astrocytes while also diminishing disease severity in EAE in conjunction with suppression of neuroinflammation.

Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up-or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up-or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.

>Magister Scientiae - MSc / Purpose: One of the leading causes of death reported in women worldwide is breast cancer. Manytumours, including breast cancer, associated with poor prognosis, have received a renewed focus and increased perspective with regard to drug discovery and innovation towards developing rational combination regimens of first-line anticancer drugs with novel compounds that target diverse hallmarks of the cancer phenotype. Multidrug resistance (MDR), which has been found to significantly decrease the efficacy of anticancer drugs and causes tumor recurrence, has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades. Several mechanisms of overcoming drug resistance have been postulated and the well known P-glycoprotein (P-gp) including other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells which in turn results in unsuccessful chemotherapy treatments. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesizes proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Previous studies demonstrated that geldanamycin (GA), a benzoquinone ansamycin antibiotic, the antibiotic, tunicamycin (TM) and the sesquiterpene lactone, thapsigargin (TG) have been found to cause ER stress and consequently, cellular arrest. GA is known to manifest anti-cancer activity through the inhibition of Hsp90-chaperone, TM interferes with N-glycosylation of newly synthesized proteins triggering the unfolded protein response, while TG inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Cellular stress conditions, lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen which results in a unfolded protein response (UPR) to maintain cell survival in cancer cells. ERS has been previously reported to enhance MDR1 transcriptional induction and P-gp transport function in cancer cells, however, prolonged endoplasmic reticulum stress conditions and inadequate unfolded protein response force cells undergo apoptosis. In this study, we examined the effects of GA, TG and TM alone and in combination to determine the cellular response of the MCF-7 breast carcinoma cell line with regard to proliferation and P-gp-mediated drug efflux activity and apoptosis. Methods: Analyses of MCF-7 breast carcinoma cells exposed to Endoplasmic Reticulum Stress (ERS) inducers geldanamycin, thapsigargin and tunicamycin, alone and in combination, included growth curves alone and in the presence of 24 hour IC50 inhibitory concentrations of the 3 ERS inducers alone, dose-response curves (MTT cytotoxicity assays) of the ERS alone and in combination, analysis of P-glycoprotein-mediated efflux pump activity in the presence of the ERS inducers alone and in combination (Calcein-AM efflux assays), analysis of viability, cytotoxicity and early apoptosis via caspase-3/7 expression (Triplex assay) and morphological staining of apoptotic and/or necrotic cells in the presence of IC50 inhibitory concentrations of the ERS inducers alone with Annexin V-FITC. Results: This study investigated the effects of Endoplasmic Reticulum Stress (ERS) inducers on growth and proliferation of MCF-7 breast carcinoma cells in culture. The MCF-7 cell line was exposed to different concentrations of ERS inducers alone and in combination with each other. All responses occurred in a dose- and time- dependent manner. When combined at equimolar log dose concentrations, integrated effects yielded enhanced cytotoxic properties as IC50 values were drastically decreased in combination as opposed to single ERS inducer responses. Combined effect on P-glycoprotein-mediated drug efflux activity yielded minor but insignificant decreases in efflux pump activity at different time intervals as opposed to the increase in cellular efflux in the presence of the ERS inducers alone at different time intervals. Caspase-3/7 apoptotic protein expression was increased as log doses of ERS inducers alone were increased, leading to cell necrosis at higher cytotoxic concentrations. The determined IC50 growth inhibitory concentrations after 24 hours were confirmed by the Annexin V-FITC demonstrating early apoptotic, necrotic and viable cells in the presence of the ERS inducers alone. Conclusion: This study demonstrated a significant growth inhibition of MCF-7 breast carcinoma cells upon exposure to ERS inducers alone. Results suggested that when ERS inducers are used in combination, their efficacy is enhanced as 50 percent inhibitory concentrations were considerably lower in combination as opposed to when used alone. The present study is consistent with previous studies with geldanamycin, and was the 1st to investigate the effects of geldanamycin, thapsigargin and tunicamycin in combination and with reference to P-gp efflux activity. Results suggested that in combination, efflux activity may be reduced, and efficacy may be enhanced. To enhance efficacy would be a major breakthrough in cancer drug discovery and development-targeting specific populations of cancer cells and reducing ERS-induced toxicity to normal cells and vital organs.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13235号 / 論医博第2175号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 渡邊 直樹, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)