MLKL-매개 내막수송 결함에 의한 TRAIL-유도 암세포 사멸신호 증가에 대한 연구
Perturbation of MLKL-mediated endosomal trafficking enhances TRAIL signal to increase cancer cell death
- 주제(키워드) MLKL , TRAIL , Endosomal trafficking
- 발행기관 아주대학교
- 지도교수 김유선
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029194
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Mixed Lineage Kinase Domain-like (MLKL) is the essential molecule of programmed necrotic cell death, Necroptosis. To induce necroptosis, MLKL is phosphorylated by upstream partner, Receptor Interacting Protein Kinase-3 (RIPK3 or RIP3) and then translocate cellular membrane to disrupt membrane integrity. Recent data suggests that function of MLKL in RIP3-indendent manner in the effective generation of intraluminal and extracellular vesicles as well as in myelin sheath breakdown to promotes sciatic nerve regeneration. In this study, we investigated that whether MLKL play a role in link between TRAIL receptor endocytosis and apoptosis. Depletion of MLKL enhances TRAIL-induced cell death in various cancer cell lines and enhancement of cell death is RIP3-independent manner. TRAIL-induced cytotoxic signals are further increased by preventing endocytosis and MLKL depletion facilitates degradation of TRAIL-DR5. TRAIL-induced intracellular downstream signals were prolonged by depletion of MLKL and then prolonged cytotoxic signals promote cell death. Our data indicate that defect of the intracellular trafficking of TRAIL-DR5 by depletion of MLKL enhances cancer cell death. Based on our findings, it is tempting to speculate that to promote death receptor signals, it may effective to reduced MLKL expression and to prevent survival receptor signal, upregulated MLKL expression contribute to abolish the intracellular signal through the MLKL-dependent extracellular vesicles secretion. Although the processes that regulate these pathways require further elucidation for detail molecular mechanisms, both ways will be the potent therapeutic strategies to target cancer.
more목차
ABSTRACT -- i
TABLE OF CONTENTS -- iii
LIST OF FIGURES -- V
I. INTRODUCTION -- 1
II. MATERIALS AND METHODS -- 4
A. GST-TRAIL purification -- 4
B. Antibodies and chemical reagents -- 5
C. Cell culture -- 5
D. Lentiviral shRNA experiments -- 6
E. Cytotoxicity assay -- 6
F. Immunoblot analysis -- 6
G. Ligands and Receptor uptake assays, and Immunofluorescence staining -- 6
H. Flow cytometry analysis -- 7
I. GST-pull down assay -- 7
J. Reverse transcription-PCR -- 8
III. RESULTS -- 9
A. Depletion of MLKL accelerates TRAIL-induced cancer cell death -- 9
B. RIP3-independent enhancement of cell death by MLKL depletion in TRAIL-mediated cytotoxicity -- 13
C. Depletion of MLKL caused defect on receptor-ligand endosomal trafficking --21
D. Endosomal trafficking is associated with MLKL-mediated sensitization of TRAIL-cytotoxicity -- 28
E. Prolonged death signal due to the defect of MLKL-mediated endosomal trafficking -- 33
IV. DISCUSSION -- 36
V. REFERENCES -- 38
국문요약 -- 43
