碩士 / 中國醫藥大學 / 生物科技學系碩士班 / 99 / Deguelin, is a natural product, isolated from Derris trifoliata Lour. It was
reported that deguelin inhibited cell proliferation and caused cell death in human
leukemia and breast cancer cells through the induction of apoptosis. Therefore, we
investigate deguelin whether or not affect human lung cancer cells in vitro . First,
we observed the change of cell morphology and percentage of cell viability after
various doses of deguelin treatment, flow cytometric analysis and trypan blue
staining were used to detect the cell viability and the cell growth inhibition of
NCI-H460 cells. Second, we used annexin V, DAPI staining, comet assay and DNA
gel electrophoresis to determined the phenomenon of apoptosis. Finally, we
investigated signal transduction by examining the intracellular change of
mitochondria membrane potential (Δψm) and Ca2+ levels, western blotting, caspase
activity, AKT kinase assay and immunofluoresence were used in NCI-H460 cell
line. Based on these results, we found that deguelin greatly suppressed cell
proliferation and decreased cell viability in time- and dose-dependent manners.
Deguelin induced cell apoptosis and DNA damage including chromatin
condensation, the formation of apoptotic bodies, and translocation of
phosphatidylserine (PS) of the plasma membrane. Western blotting and
immunofluoresence proved that deguelin inhibited p-AKT protein expressions,
resulting in activation of Bad and then translocate from cytosol to mitochondria
where it binds to Bcl-2 and Bcl-X, followed by promoted the influence of Bcl-2
family proteins. There is a decrease in mitochondria membrane potential (Δψm),
causing release of cytochrome c and AIF. Cytochrome c combined with apaf-1 and
caspase-9 to an apoptosome , activated caspase-3, leading to apoptosis. Then, AIF
translocated into nuclear causing DNA damage. Overall, we suggest that the major
mechanism of deguelin-induced apoptosis in NCI-H460 is through AKT and
Bad-releated signaling pathway.
| Identifer | oai:union.ndltd.org:TW/099CMCH5111006 |
| Date | January 2011 |
| Creators | Yu-chieh Hsu, 徐于絜 |
| Contributors | Chung- Jing-Gung, 鍾景光 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 60 |
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