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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌(Po-Huang Liang) | |
| dc.contributor.author | Jhih-Jhong Dong | en |
| dc.contributor.author | 董至中 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:43:10Z | - |
| dc.date.available | 2012-07-18 | |
| dc.date.copyright | 2011-07-18 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-06 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47934 | - |
| dc.description.abstract | 先前我們合成了一化合物Iodoacetyl-Boc-lysine (I-Lys)並確認其在MCF-7乳癌細胞中，藉由直接活化caspase-7引發細胞凋亡的能力。在這篇論文中，我們試圖用X光結晶學、二維核磁共振以及液態層析質譜儀來確認I-Lys在caspase-7上的結合位置，雖然X光結晶學以及二維核磁共振的方式失敗，我們還是用液態層析質譜儀確認了caspase-7的半胱氨酸246為I-Lys的結合位置，藉由在MCF-7細胞中強迫表達C246S 突變的caspase-7，我們確認了I-Lys在MCF-7細胞中對半胱氨酸246的專一性，我們也藉由staurosporine以及caspase活性測試確認了C246S 突變的caspase-7的酵素仍具活性。而藉由即時測定細胞內caspase-7活性，我們發現I-Lys的加入會立即提升細胞內caspase-7的活性且此活性並非來自procaspase-7活化造成的活化型caspase-7總量上升。XIAP:caspase-7複合體的免疫沉澱證明了I-Lys專一的標定p19/p12-casp7並破壞XIAP: p19/p12-casp7複合體以引發MCF-7的細胞凋亡，在穩定表達D23A突變的caspase-7的MCF-7細胞中，p19/p12-casp7的數量減少指出了p19/p12-casp7在MCF-7中會持續存在。從一些癌症和正常細胞，我們發現XIAP:p19/p12-casp7只有表現在caspase-3不表達之乳癌細胞，給予了在對付caspase-3不表達的乳癌細胞時，以XIAP:p19/p12-casp7為標靶的專一性。最後，藉由在MCF-7細胞中重建caspase-3的表達，我們發現表達caspase-3的MCF-7對I-Lys具抵抗力並因此確定I-Lys對caspase-3不表達之乳癌細胞的專一性。
總括而論，這些研究發現了I-Lys的標的以及在MCF-7細胞中持續堆積的XIAP:p19/p12-casp7複合體，說明了I-Lys在MCF-7細胞中引發細胞凋亡的機制，並且提出一個對臨床上caspase-3表達量下降的乳癌細胞的治療標靶 | zh_TW |
| dc.description.abstract | Previously, from an iodoacetamide-based compound library, iodoacetyl-Boc-lysine (I-Lys) was identified to induce cell apoptosis in MCF-7 breast cancer cell line by directly activating caspase-7. In this thesis, I attempted to use X-ray crystallography, 2D NMR, and LC-mass to identify the binding site of I-Lys on caspase-7. We identify Cys246 residue as a targeting site of I-Lys in caspase-7 by LC-MS although the studies of X-ray crystallography and 2D NMR failed. Then we showed the targeting specificity of I-Lys on Cys246 in vivo by enforcedly expressing caspase-7 C246S mutation in MCF-7 cells. We also showed that caspase-7 C246S mutant still possess the activity by using staurosporine and caspase activity assay. By determining real-time caspase-7 activity in cells, we found that I-Lys immediately elevates intracellular caspase-7 activity without increaseing the active-form caspase-7 level by procaspase-7 activation/processing in MCF-7 cells . Immunoprecipitation of XIAP:caspase-7 complex proved that I-Lys specifically targets p19/p12-casp7 to induce apoptosis of MCF-7 cells by disrupting the XIAP: p19/p12-casp7 complex. The reduction of p19/p12-casp7 level in MCF-7 cells stably expressing caspase-7 D23A mutant indicates that the production of p19/p12-casp7 constitutively occur in MCF-7 cells. From a panel of cancerous and normal cells, we found exclusive deposition of XIAP:p19/p12-casp7 complex in caspase-3null breast tumors, conferring the specificity of I-Lys in killing those tumors over other caspase-3-expressing cells in vitro and in vivo. Finally, by reconstituting caspase-3 expression into MCF-7 cells, we found that MCF-7/CASP3 cells are resistant to I-Lys and therefore confirmed the specificity of I-Lys to caspase-3null breast tumors.
Overall, these studies find out the target of I-Lys and the constitutively deposited XIAP:p19/p12-casp7 complexes in MCF-7 cells, elucidate the mechanism of I-Lys to induce cell apoptosis in MCF-7 cells and to suggest a target of cancer treatment for caspase-3 down regulated breast tumors in clinical. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:43:10Z (GMT). No. of bitstreams: 1 ntu-100-R98b46016-1.pdf: 3922042 bytes, checksum: 6a1eee65e7f2236df6bdb2e24f28188d (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………………………………….….1
ABSTRACT……………………………………………………………………………………….…2 (1)INTRODUCTION……………………………………………………………………………….4 1.1 Treatments for breast tumor………………………………………………………...…......4 1.2 Caspase family and their functions in apoptosis…………………………………………..6 1.3 X-linked Inhibitor of apoptosis protein (XIAP)…………………………………………...8 1.4 Previous studies and present works………………………………………………………..9 (2)MATERIALS AND METHODS……………………………………………………………….11 2.1 Reagents…………………………………………………………………………………..11 2.2 Flow cytometric analysis…………………………………………...………………….....11 2.3 Determination of caspase activity………………………………………………………...11 2.4 Immunoprecipitation……………………………………………………………………..12 2.5 Protein expression and purification……………………………………………………….12 2.6 Stable cell line generation………………………………………………………………..13 2.7 Identification of I-Lys-alkylated Cys residue(s) in caspase-7 by LC-MS………………...13 2.8 Western blot analysis……………………………………………………………………..14 2.9 Crystallization and data collection……………………………………………………….14 2.10 2D 15N TROSY-HSQC spectrum………………………………………………………...15 (3)RESULTS………………………………………………………………………………………..16 3.1 I-Lys incorporates into Cys246 residue of caspase-7……………………………………...16 3.1.1 Identifying the targeted cysteine residue(s) by X-ray crystallography………...16 3.1.2 Identifying the targeted cysteine residue(s) by 2D NMR……………………...16 3.1.3 Identifying the targeted cysteine residue(s) by liquid chromatography -mass spectrometry………………………………………………………………………...17 3.1.4 The stable cell line of MCF-7 overexpress caspase-7C246S is resistant to I-Lys.18 3.1.5 Staurosporine induces cell apoptosis in both wild-type MCF-7 and MCF-7/casp7C246S stable cell line…………………………………………………...19 3.1.6 I-Lys induces the activation of caspase-7 only in wild type MCF-7, but staurosporine induces in both wild type and casp7C246S stable cell line…………….19 3.2 I-Lys increases the activity of caspase-7 within seconds but not the amount of active-form caspase-7 within 24hr in MCF-7 cell line………………………………………………..20 3.2.1 I-Lys increases the activity of caspase-7 within seconds but not the amount of active-form caspase-7 within 24hr in MCF-7 cell line……………………………...20 3.2.2 I-Lys impairs the interaction between caspase-7 and X-linked inhibitor of apoptosis protein (XIAP) in MCF-7 cell line……………………………………….21 3.2.3 I-Lys induces cell apoptosis in MCF-7/casp7D23A stable cell line at higher dose than wild type MCF-7 and MCF-7/casp7wt…………………………………………22 3.2.4 Staurosporin can still induce cell apoptosis in MCF-7/casp7D23A stable cell line…………………………………………………………………………………..22 3.3 I-Lys selectively causes cell apoptosis in caspase-3-deficient tumors…………………...23 (4)DISCUSSION…………………………………………………………………………………...25 REFERENCE………………………………………………………………………………….......28 TABLE…………………………………………………………………………………...................34 FIGURE…………………………………………………………………………………………….35 SUPPLEMENTARY……………………………………………………………………………….52 APPENDIX…………………………………………………………………………………...........59 | |
| dc.language.iso | en | |
| dc.subject | 乳癌 | zh_TW |
| dc.subject | breast cancer | en |
| dc.subject | MCF-7 | en |
| dc.subject | XIAP | en |
| dc.subject | caspase-7 | en |
| dc.subject | I-Lys | en |
| dc.title | 探討MCF-7乳癌細胞中常態堆積之XIAP:p19/p12-Casp7複合體作為I-Lys標靶 | zh_TW |
| dc.title | Studying on the constitutively deposited XIAP:p19/p12-casp7 complexes in MCF-7 breast cancer cells as a target for I-Lys | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡明道(Ming-Daw Tsai),陳瑞華(Ruey-Hwa Chen) | |
| dc.subject.keyword | 乳癌, | zh_TW |
| dc.subject.keyword | breast cancer,caspase-7,XIAP,MCF-7,I-Lys, | en |
| dc.relation.page | 61 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-06 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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