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Graduate School of Ajou University Department of Molecular Science and Technology 3. Theses(Master)

세포사멸수용체 4 (DR4) 특이 항체의 세포사멸 기전 및 항암 소분자와의 병합처리에 의한 암세포사멸 증진효과 기작 연구

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dc.contributor.advisor김용성-
dc.contributor.authorSung, Eun-Sil-
dc.date.accessioned2018-11-08T07:56:11Z-
dc.date.available2018-11-08T07:56:11Z-
dc.date.issued2013-08-
dc.identifier.other14754-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/8617-
dc.description학위논문(박사)아주대학교 일반대학원 :분자과학기술학과,2013. 8-
dc.description.tableofcontentsCONTENTS …………………………………………………..………… 1 ABSTRACT OF DISSERTATION …………………………………… 6 LIST OF TABLES ................................................................... 9 LIST OF FIGURES ……………………………...………………..…... 10 CHAPTER 1. General introduction ………………………………… 13 1.1. Death receptor (DR) signaling ……………………………….… 13 1.2. Agonistic antibodies to DRs as cancer therapeutics ……………… 16 1.3. Combined Trials of TRAIL and Agonistic antibodies to DRs …… 17 CHAPTER 2. A novel agonistic antibody to human DR4 induces apoptotic cell death in various tumor cells ……………………..……. 20 2.1 Abstract ………………………………………..……..…………… 20 2.2 Introduction …..…………………………….……………………... 22 2.3 Materials and Methods …………………………..……………...… 25 2.3.1 Generation and Characterization of AY4 mAb against Human DR4 …………………...…...…………………………...………...… 25 2.3.2 Reagents …………………………………………..….………. 25 2.3.3 Cell lines and Cell viability assays …………………………… 26 2.3.4 Western blotting analysis ……………………………………... 27 2.3.5 Analysis of tumoricidal activity In vivo ……………………… 27 2.4 Results ……………………………..……………………………… 29 2.4.1 AY4 specifically binds to DR4 without competition with TRAIL …………….…...…...………………………...…………....… 29 2.4.2 AY4 alone induces cell death of various cancer cell lines without cytotoxicity in normal human hepatocytes …….……...…..….. 34 2.4.3 AY4 induces caspase-dependent apoptotic cell death .......…… 43 2.4.4 FADD and caspase-8 are essential for the AY4-mediated cell death …………..………..…………………………………..…….…. 47 2.4.5 AY4 activates both extrinsic and intrinsic apoptotic pathway …………………………..…………………………………….. 50 2.4.6 AY4 exhibits antitumor activity In vivo …………..………… 52 2.5 Discussion …………………………………..………………………. 54 CHAPTER 3. HDAC inhibitors synergistically potentiate DR4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cell ………………………………………………………………………...… 59 3.1 Abstract ………………………………………………………….. 59 3.2 Introduction ……...……………………………………………… 60 3.3 Material and Methods ……………………………………………. 64 3.3.1 Materials ……………………………..……………………… 64 3.3.2 Cell lines and cell cultures …………………..……………… 64 3.3.3 Cell viability assays ………………………..………………... 65 3.3.4 Western blotting analysis ……………………..……………... 70 3.3.5 Flow cytometry ……………………………..……………… 70 3.3.6 RNA interference ……………………………..……………… 70 3.3.7 Statistical analysis …………………………………..………. 71 3.4 Results …………………………………………………………… 72 3.4.1 Responses of various leukemia cell lines to AY4 or TRAIL alones ......................................................................................... 72 3.4.2 Screening of chemotherapeutic agents for synergistic effects on TRAIL-mediated cytotoxicity in various leukemia cell lines … 75 3.4.3 SAHA and VPA synergize both AY4- and TRAIL-induced apoptosis ………………………………………………...…… 77 3.4.4 Cell surface expression levels of DR4 and DR5 are not closely correlated with AY4 or TRAIL-induced cytotoxicity ……….. 80 3.4.5 SAHA and VPA synergize with both AY4- and TRAIL-mediated cell death by amplifying both extrinsic and intrinsic apoptotic pathways …………………………..………………………….. 83 3.4.6 Combined treatment of AY4 or TRAIL with either SAHA or VPA downregulates expression level of c-FLIP …………………... 86 3.4.7 Combined treatment of AY4 or TRAIL with either SAHA or VPA downregulates expression levels of anti-apoptotic proteins ….. 90 3.4.8 Combined treatment of AY4 with the other agents does not significantly affect expression level of anti-apoptotic proteins ……………………………………………………………….. 92 3.5 Discussion ………………………………………..……………….. 93 CHAPTER 4. The proteasome inhibitor potentiates TRAIL receptor agonist-induced apoptosis by stabilizing tBid and Bik in human head and neck squamous cell carcinoma cells …………………………….. 100 4.1 Abstract ……………………………..……………………..…….. 100 4.2 Introduction ………………………….…………………………... 102 4.3 Materials and Methods ………………………………………… 105 4.3.1 Cell lines and reagents …………………………………... 105 4.3.2 Cell treatment and viability assay ………………………. 106 4.3.3 Synergistic analysis of combined treatments ……………. 107 4.3.4 Analysis of cell surface expression of TRAIL receptors … 107 4.3.5 Western blotting and immunoprecipitation ……………… 107 4.3.6 Subcellular fractionation …………………………..……... 108 4.3.7 RNA interference …………………………………..……... 108 4.3.8 Protein stability assay ……………………………..……… 109 4.3.9 Statistical analysis ……………………………….….………. 109 4.4 Results …………………………………………………………… 110 4.4.1 MG132 sensitizes HNSCC cells to TRAIL-/AY4-mediated cell death by activating extrinsic and intrinsic apoptotic pathways .. 110 4.4.2 MG132 significantly increases DR5 surface expression and induces accumulation of proapoptotic Bik and antiapoptotic Mcl-1.. 116 4.4.3 MG132-mediated stabilization of Bik and tBid contributes to the synergistic cell death induced by combination treatment ….. 119 4.4.4 Bak, but not Bax, is constrained by Mcl-1 and Bcl-XL and released by Bik accumulated due to MG132 …………………...…….. 124 4.4.5 Knockdown of Mcl-1 and Bcl-XL significantly sensitizes HNSCC cells to TRAIL and AY4 as single agents ….………………... 127 4.4.6 Combination treatment induces translocation of tBid and Bax from the cytosol to the mitochondria …………………………...… 129 4.5 Discussion ………………………………………………….……. 133 CONCLUSION ……………………………………………………… 138 REFERENCES ……………………………………………………… 140 ABSTRACT IN KOREAN ………………………………………..... 155 PUBLICATIONS ……………………………………………………. 159-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title세포사멸수용체 4 (DR4) 특이 항체의 세포사멸 기전 및 항암 소분자와의 병합처리에 의한 암세포사멸 증진효과 기작 연구-
dc.title.alternativeEun-Sil Sung-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameEun-Sil Sung-
dc.contributor.department일반대학원 분자과학기술학과-
dc.date.awarded2013. 8-
dc.description.degreeMaster-
dc.identifier.localId571232-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000014754-
dc.description.alternativeAbstractIdeal anti-cancer therapeutics would induce tumor cell death selectively but not normal cells. In this regard, pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5, TRAIL-R2), are attractive targets for the development of agonistic anti-cancer biologics. Recently, several agonistic monoclonal antibodies (mAb) have been developed by targeting DR4 or DR5, which induce cell death in various types of tumor in vitro and in vivo. Some of them are now under various phases of clinical trials. However, some tumor cells are resistant to TRAIL-induced apoptosis. This resistance can be overcome by its combination with anti-cancer chemotherapeutic agents [1-3]. Here I report the cell death mechanism of a mouse agonistic antibody against human DR4 and its synergistic anti-cancer mechanism in combination with anti-cancer agents in various tumor types. In chapter 1, general introduction was described about Death Receptor (DR)-mediated signaling, developments of agonistic antibodies to DRs, and its combination therapy with anti-cancer agents. In chapter 2, I report about characterization of a novel agonistic antibody to human death receptor 4 in various tumor cells. An agonistic mAb, AY4, raised against human DR4 in mice, which did not compete with DR4-TRAIL interaction. The anti-DR4 agonistic AY4 mAb induced caspase-dependent apoptotic cell death in various types of cancer cells without significant toxicity to normal human hepatocytes. AY4 reduced the growth of pre-established human lung tumors in a mouse xenograft model. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which were only sensitive to TRAIL and anti-DR5 agonistic mAbs but resistant to DR4-selective TRAIL mutants and other anti-DR4 agonist mAbs [4, 5]. In chapter 3, I investigated that HDACI (Histone deacetylase inhibitors) have synergistic effects on AY4-mediated cell death in the T-cell acute lymphoblastic leukemia (T-ALL) cells. In this study, I investigate the specific role of DR4 in inducing apoptosis by studying the susceptibility of various leukemia cell lines to AY4 alone or in combination with various chemotherapeutic agents, in comparison with TRAIL. I found that most leukemia cell lines are intrinsically resistant to AY4 or TRAIL alone, but two T-ALL cell lines (CEM-CM3, CCRF-CEM) become substantially sensitized by combined treatment of AY4 or TRAIL with HDACI, either SAHA or VPA, by synergistic amplification of both the extrinsic and intrinsic apoptotic pathways. All of the combined treatments synergistically downregulated the expression of several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-XL, XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. These results demonstrate that the HDACIs have synergistic effects on AY4-mdiated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells. In chapter 4, I investigated the effects of the proteasome inhibitor MG132 in combination with TRAIL or AY4 on sensitization of TRAIL- and AY4-resistant human HNSCC (head and neck squamous cell carcinoma cells) cell lines. This study provides a novel mechanism for the synergistic apoptotic cell death, which involves the generation and stabilization of tBid and the stabilization of Bik. These results indicate that tBid directly activates Bax, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-XL, suggesting a major role for the mitochondrial apoptotic pathway in the synergistic apoptotic cell death mediated by combination treatment in HNSCC cells. Moreover, restriction of Bak to the mitochondria by Mcl-1 and Bcl-XL is an important mechanism of resistance to DR5-/DR4-mediated apoptotic cell death in HNSCC cells. The potent synergy between the proteasome inhibitor and TRAIL receptor agonists suggests that the combination of the two agents may offer a new therapeutic strategy to improve HNSCC treatment. In conclusion, I studied cell death mechanism of an agonistic anti-DR4 antibody in various tumor types. However, it has resistance to TRAIL or AY4 in many tumor cells, especially, T-ALL and HNSCC cells. To overcome the resistance, I evaluated AY4 in combination with various therapeutic agents to compare TRAIL-mediated combination effects. Here, I studied AY4-mediated cell death and molecular mechanism in treatment with the combination of various anti-cancer agents with TRAIL or AY4 in most of which are completely resistant to TRAIL and AY4 as single agents. These results suggest that the combination with anti-DR4 agonistic mAb AY4 and general anti-cancer agents can be effective in resistant tumor cells.-
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