Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells

碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 100 === Numerous studies have demonstrated the anticancer effect of triterpenoids. A previous study from our lab has shown that Dehydroeburicoic acid (DeEA), a triterpenoid isolated from Antrodia cinnamomea (known as “niu-chang-chin” in Taiwan), can induce necr...

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Main Authors: Jun-Liang Kuo, 郭峻良
Other Authors: Chung-Jiuan Jeng
Format: Others
Language:zh-TW
Published: 2012
Online Access:http://ndltd.ncl.edu.tw/handle/85290205231360875476
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spelling ndltd-TW-100YM0053910172015-10-13T21:22:40Z http://ndltd.ncl.edu.tw/handle/85290205231360875476 Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells 去氫齒孔酸及其衍生物導致神經膠原瘤細胞死亡的作用機轉 Jun-Liang Kuo 郭峻良 碩士 國立陽明大學 解剖學及細胞生物學研究所 100 Numerous studies have demonstrated the anticancer effect of triterpenoids. A previous study from our lab has shown that Dehydroeburicoic acid (DeEA), a triterpenoid isolated from Antrodia cinnamomea (known as “niu-chang-chin” in Taiwan), can induce necrotic cell death in the human glioblastoma cell U87MG via Ca2+ overload, mitochondrial dysfunction, and calpain activation. In this thesis, we aim to further examine the mechanisms of DeEA-induced cell death in U87MG cells. In addition, we explored the anti-proliferative effects of two DeEA-derived compounds, Methyl Dehydroeburicoate (M-DeEA) and Dehydroeburicoic acid monoacetate (MA-DeEA). Cell viability and cytotoxicity assays indicated that all three compounds can inhibit the proliferation of U87MG cells. Light microscopic analyses showed that the three compounds display differential morphological effects: DeEA induced shortening round-up and cell rupture; M-DeEA also induced cell shortenting, but with no significant rupture; and MA-DeEA induced irregular cell elongation or floating round-up. We therefore moved on to examine the differential anti-proliferation and cell death mechanisms of the three compounds. Flow cytometric analyses with Annexin-V and propidium iodide labeling showed that DeEA significantly enhanced necrotic cell death, but that both M-DeEA and MA-DeEA moderately increased apoptotic cell death. The results of AO/EtBr staining assay also supported the idea that M-DeEA induced apoptosis. Pre-treatment with the caspase inhibitor zVAD-fmk, however, failed to significantly block MA-DeEA-induced apoptotic cell death. On the other hand, cell cycle analyses with PI labeling suggested that MA-DeEA induced cell cycle arrest at the G2/M phase. Immunofluoresence staining of LC3B showed that DeEA significantly enhanced the presence of autophagosomes, Examination of the mitochondrial membrane potential with Mitotracker demonstrated that both DeEA and MA-DeEA disrupted the function of mitochondria, which in turn may induce cell death. Immunofluoresence staining of PARP and AIF further showed that both DeEA and MA-DeEA induced the translocation of AIF from mitochondria to nucleus, and that DeEA notably enhanced the expression of PARP. In summary, we have identified differential cell death effects in U87MG cells by DeEA, M-DeEA, and MA-DeEA: DeEA causes PARP/AIF-related necrosis; M-DeEA triggers apoptotic cell death; and MA-DeEA probably induces cell cycle arrest at the G2/M phase. Chung-Jiuan Jeng 鄭瓊娟 2012 學位論文 ; thesis 67 zh-TW
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language zh-TW
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description 碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 100 === Numerous studies have demonstrated the anticancer effect of triterpenoids. A previous study from our lab has shown that Dehydroeburicoic acid (DeEA), a triterpenoid isolated from Antrodia cinnamomea (known as “niu-chang-chin” in Taiwan), can induce necrotic cell death in the human glioblastoma cell U87MG via Ca2+ overload, mitochondrial dysfunction, and calpain activation. In this thesis, we aim to further examine the mechanisms of DeEA-induced cell death in U87MG cells. In addition, we explored the anti-proliferative effects of two DeEA-derived compounds, Methyl Dehydroeburicoate (M-DeEA) and Dehydroeburicoic acid monoacetate (MA-DeEA). Cell viability and cytotoxicity assays indicated that all three compounds can inhibit the proliferation of U87MG cells. Light microscopic analyses showed that the three compounds display differential morphological effects: DeEA induced shortening round-up and cell rupture; M-DeEA also induced cell shortenting, but with no significant rupture; and MA-DeEA induced irregular cell elongation or floating round-up. We therefore moved on to examine the differential anti-proliferation and cell death mechanisms of the three compounds. Flow cytometric analyses with Annexin-V and propidium iodide labeling showed that DeEA significantly enhanced necrotic cell death, but that both M-DeEA and MA-DeEA moderately increased apoptotic cell death. The results of AO/EtBr staining assay also supported the idea that M-DeEA induced apoptosis. Pre-treatment with the caspase inhibitor zVAD-fmk, however, failed to significantly block MA-DeEA-induced apoptotic cell death. On the other hand, cell cycle analyses with PI labeling suggested that MA-DeEA induced cell cycle arrest at the G2/M phase. Immunofluoresence staining of LC3B showed that DeEA significantly enhanced the presence of autophagosomes, Examination of the mitochondrial membrane potential with Mitotracker demonstrated that both DeEA and MA-DeEA disrupted the function of mitochondria, which in turn may induce cell death. Immunofluoresence staining of PARP and AIF further showed that both DeEA and MA-DeEA induced the translocation of AIF from mitochondria to nucleus, and that DeEA notably enhanced the expression of PARP. In summary, we have identified differential cell death effects in U87MG cells by DeEA, M-DeEA, and MA-DeEA: DeEA causes PARP/AIF-related necrosis; M-DeEA triggers apoptotic cell death; and MA-DeEA probably induces cell cycle arrest at the G2/M phase.
author2 Chung-Jiuan Jeng
author_facet Chung-Jiuan Jeng
Jun-Liang Kuo
郭峻良
author Jun-Liang Kuo
郭峻良
spellingShingle Jun-Liang Kuo
郭峻良
Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
author_sort Jun-Liang Kuo
title Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
title_short Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
title_full Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
title_fullStr Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
title_full_unstemmed Mechanisms Underlying Dehydroeburicoic Acid Derivatives-induced Cell Death in U87MG Glioblastoma Cells
title_sort mechanisms underlying dehydroeburicoic acid derivatives-induced cell death in u87mg glioblastoma cells
publishDate 2012
url http://ndltd.ncl.edu.tw/handle/85290205231360875476
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