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Title: | 蛋白解體抑制劑引發基因轉錄與細胞凋亡之探討 Regulatory mechanism of proteasome inhibitors-induced gene transcription and apoptosis |
Authors: | Jun-Jie Chen 陳俊杰 |
Advisor: | 陳青周 |
Keyword: | 蛋白解體抑制劑,環氧化酶,-2,死亡受體-5, Proteasome inhibitor,cyclooxygenase-2,death receptor-5, |
Publication Year : | 2007 |
Degree: | 博士 |
Abstract: | 抑制泛素-蛋白解體路徑有希望成為治療發炎及癌症的明日之星,蛋白解體抑制劑MG132除了可透過泛素-蛋白解體路徑調節蛋白轉化作用外,也被報導可經由mitogenic activated kinases (MAPKs)活化activating protein (AP-1),並引發許多發炎基因如monocyte chemoattractant protein-1(MCP-1)、IL-8及IL-6。然而,蛋白解體抑制劑啟動發炎基因的詳細機制仍未完全清楚。在本論文中,我們發現在人類肺泡上皮癌細胞NCI-H292、A549及胃上皮癌細胞AGS中,蛋白解體抑制劑MG132、PSI-1及lactacystin引發COX-2表現是透過增加基因轉錄而非阻止蛋白分解的機制而來。COX-2 promoter上NF-IL6與CRE結合位參與此基因轉錄是必須的,而NF-kB結合位則不參與此機制。CCAAT/enhancer結合蛋白(C/EBPb)和C/EBPd結合到NF-IL6和CRE位置,並且MG132可增加組蛋白H3和H4乙醯化並引發CBP徵召到COX-2 promoter上。然而,C/EBPb和C/EBPd之蛋白質總量則不受MG132改變。MG132所引發C/EBPd而非C/EBPb之DNA結合活性是透過p38、PI3K、Src和PKC所調節的。C/EBPd之小片段干擾RNA (siRNA)抑制COX-2表現進一步證明C/EBPd在COX-2基因轉錄的角色。此外,MAPKs及Akt參與MG132刺激細胞內反應性氧化物種(ROS)之生成。這些發現顯示蛋白解體抑制劑引發COX-2轉錄必須透過ROS-dependent蛋白激酶的活化及之後C/EBPd和CBP之徵召。
在非小細胞肺癌中,蛋白解體抑制劑PS-341可致敏TRAIL媒介之細胞凋亡。MG132已被報導可透過CHOP的增加而引發DR5。Lactacystin在人類神經膠細胞中可增進Fas媒介之細胞凋亡,這些研究顯示蛋白解體抑制劑可透過增加死亡受器的表現而誘導細胞凋亡,然而其詳細機轉仍然未知。 以調節TRAIL受器-2 (DR5)路徑作為標的有希望成為根除癌細胞之治療策略,臨床研究顯示蛋白解體抑制劑增進TRAIL引發之細胞凋亡,但其機轉仍未完全了解。本論文中,我們發現在肺部上皮細胞中蛋白解體抑制劑致敏TRAIL媒介之細胞凋亡是透過引發DR5表現而來。Reporter assay顯示在 DR5第一intron區域的p53和NF-kB位置參與此過程是必須的。p53 siRNA和NF-kB抑制劑可壓制DR5表現,更進一步證實了p53和NF-kB在DR5轉錄中所扮演的角色。我們也發現MG132可增加p53表現和穩定度、Ser392磷酸化及Lys373/382乙醯化。同時,可觀察到IkBa的分解和NF-kB轉位的現象。DAPA及ChIP assay顯示p53及p65可結合到DR5第一intron的區域,MG132增加細胞內氧化活性物種(ROS)之生成,可導致p53而非p65的核轉位和DNA結合活性。ROS scavenger可明顯抑制蛋白解體抑制劑併用TRAIL所引發之細胞凋亡。這些發現顯示蛋白解體抑制劑所媒介的NF-kB和ROS-dependent p53活化參與了DR5轉錄之intronic調節作用,並増進TRAIL-所誘發之細胞凋亡。 總結,本論文對蛋白解體抑制劑在COX-2及DR5轉錄作用之調控提供詳細機轉之探討,並進一步釐清氧化活性物種及下游轉錄因子在蛋白解體抑制劑活化基因轉錄過程中所扮演的角色,而根據此論文我們可了解蛋白解體抑制劑對發炎及細胞凋亡基因之調控作用及其功能之相關性,以期望對未來蛋白解體抑制劑之藥物研究及臨床治療提供一個參考。 Inhibition of ubiquitin-proteasome pathway has been shown to be a promising strategy for the treatment of inflammation and cancer. In addition to the regulation of protein turnover via ubiquitin-proteasome pathway, the proteasome inhibitor MG132 had been reported to activate activator protein-1 (AP-1) through the mitogenic activated protein kinases (MAPKs) pathway and induce the expressions of several inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-6. However, the precise mechanism by which proteasome inhibitor triggers the expression of inflammatory genes is not fully clear. In this thesis, we show that proteasome inhibitors MG132, PSI-1 and lactacystin induce COX-2 expression via enhancing gene transcription rather than preventing protein degradation in the human alveolar NCI-H292 and A549, and gastric AGS epithelial cells. NF-IL6 and CRE, but not NF-kB elements on the COX-2 promoter were involved in the gene transcription event. The binding of CCAAT/enhancer binding protein (C/EBP)b and C/EBPd to the CRE and NF-IL6 elements, as well as the recruitment of CBP and the enhancement of histone H3 and H4 acetylation on the COX-2 promoter was enhanced by MG132. However, it did not affect the total protein levels of C/EBPb and C/EBPd. MG132-induced DNA-binding activity of C/EBPd but not C/EBPb was regulated by p38, PI3K, Src and PKC. Small interfering RNA of C/EBPd suppressed COX-2 expression, further strengthening the role of C/EBPd in COX-2 gene transcription. In addition, the generation of intracellular reactive oxygen species (ROS) in response to MG132 contributed to the activation of MAPKs and Akt. These findings reveal that the induction of COX-2 transcription induced by proteasome inhibitors requires ROS-dependent protein kinases activation and the subsequent recruitments of C/EBPd and CBP. Proteasome inhibitor PS-341 sensitized TRAIL-mediated apoptosis in non-small cell lung cancer cells. It has been reported that MG132 induced DR5 expression through CHOP up-regulation. Lactacystin enhanced Fas-mediated apoptosis by induction of Fas and Fas ligand in human glioma cells. These studies imply that proteasome inhibitors may enhance apoptosis through the up-regulation of death receptor. However, the underlying mechanism of this enhancement is still unclear. Manipulation of TRAIL receptor-2 (DR5) pathway is a promising therapeutic strategy to eradicate cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis, but the underlying mechanism has not been fully elucidated. In this thesis, we show that proteasome inhibitors sensitize TRAIL-mediated apoptosis by inducing DR5 expression in human alveolar cells. Reporter assay demonstrated the involvement of p53 and NF-kB elements on DR5 first intron region. Both p53 siRNA and NF-kB inhibitor suppressed DR5 expression, strengthening the role of p53 and NF-kB in DR5 transcription. p53 expression and stability, Ser392 phosphorylation and Lys373/382 acetylation were enhanced by MG132. Meanwhile, IkBa degradation and NF-kB translocation were also observed. DAPA and ChIP assays demonstrated the occupancy of p53 and p65 on the first intron of DR5. Intracellular reactive oxygen species (ROS) generated after MG132 treatment contributed to p53 but not p65 nuclear translocation and DNA-binding activity. ROS scavenger dramatically inhibited the apoptosis induced by proteasome inhibitors plus TRAIL. These findings reveal that proteasome inhibitors-mediated NF-kB and ROS-dependent p53 activation are attributed to intronic regulation of DR5 transcription, and resulted in the subsequent enhancement of TRAIL-induced apoptosis. Taken together, all the results provide a mechanistic insight into the crucial role of proteasome inhibitors on COX-2 and DR5 transcription, and further elucidate the role of ROS and downstream transcription factor in the process of proteasome inhibitors induced gene transcription. According to this thesis, we shed the light on the relationship between the regulation and function of inflammatory and apoptotic gene in response to proteasome inhibitors. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27501 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥理學科所 |
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