博士 / 國立臺灣大學 / 藥學研究所 / 102 / Population aging causes problems associated with age related diseases. Medical resources spending for age related diseases such as Alzheimer’s disease (AD) and cancer has increased year by year, but therapeutic options for these diseases are still limited. In this doctoral thesis, we have investigated one possible cause of Alzheimer’s disease and the potential effect of drugs targeting in the first part. In the second part, we have screened compounds especially for anti-glioblastoma and have investigated the anticancer mechanisms of these compounds.
In the first part, based on our past results in yeast system, we transfered the model from yeast to human cell lines, and determined whether we could reproduce the past results. We have found that a fragment of the transmembrane protein associated with Alzheimer’s disease, amyloid precursor protein (APP) intracellular domain (AICD), interacts with a truncated prolyl isomerase named FK506 binding protein 12 (FKBP12) by yeast-two hybrid screening, and the interaction between AICD and FKBP12 can be inhibited by FK506. In the experiments of the first part, we observed the effects of FKBP12 overexpression on APP processing and C99/ C83 production and whether FK506 can alter this effect in human cell lines overexpression APP and FKBP12. We also used APPT668E and APPT668V to mimic the phosphorylated and non-phosphorylated states of T668, testing the interactions between FKBP12 and various APP mutants by co-immunoprecipitation. The results of the first part show that FKBP12 triggered amyloidogenic pathway can be reversed by FK506.
In the second part, we have screened natural products with anti-proliferative activities against glioblastoma. We have found two natural products, calanquinone A and epi-reevesioside F, with anti-glioblastoma activity. Calanquinone A inhibited cell growth of three glioblastoma cell lines, A172, T98 and U87 cells. The data showed that calanquinone A depleted cellular glutathione. Calanquinone A induced DNA damage effect by comet assay and propidium iodide-γH2A.X double staining. N-acetyl cysteine and glutathione significantly inhibited the DNA damage effect. Furthermore, calanquinone A-mediated DNA damage was able to cause cell cycle arrest at S phase. Calanquinone A also induced the production of reactive oxygen species and an increase in intracellular Ca2+ levels. AMPK is responsible for regulating cellular energy and, therefore, a decrease in cellular ATP levels may trigger AMPK activity. As a result, calanquinone A induced glutathione-dependent AMPK activation. However, the related signal transduction pathways need further investigation.
Epi-reevesioside F showed varied effects in three glioblastoma cell lines, A172, T98 and U89. It had no effect on A172 cells but induced apoptosis in T98 cells and anti-proliferative activity in T98 and U87 cells. The addition of extracellular K+ could partly inhibited epi-reevesioside F-mediated anti-proliferative effects in T98 and U87 cells. Epi-reevesioside F induced an increase of intracellular Na+ levels, but had little effect on intracellular Ca2+ concentration. Furthermore, the increase of intracellular Na+ levels could in turn induce a decrease of cellular pH which was highly correlated with the anti-proliferative effect. In conclusion, both calanquinone A and epi-reevesioside F can inhibit cell proliferation of glioblastoma through different mechanisms. Calanquinone A decreases cellular glutathione, leading to DNA damage and S phase arrest of the cell cycle, and epi-reevesioside F induces an increase of intracellular Na+ levels, which in turn decreases intracellular pH and inhibits cell proliferation.
| Identifer | oai:union.ndltd.org:TW/102NTU05551001 |
| Date | January 2014 |
| Creators | Fan-Lun Liu, 劉凡綸 |
| Contributors | Fan-Lu Kung, 孔繁璐 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 100 |
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