TRAIL 매개 apoptosis 촉진 및 autophagic cell death를 통한 악성 glioma 선택적인 세포 사멸 유도 방안 연구
Study on the Strategies to Stimulate TRAIL-Mediated Apoptosis or Autophagic Cell Death Selectively in Malignant Glioma Cells
- 주제(키워드) Glioma , Apoptosis , Autophagic cell death , TRAIL , HDAC inhibitor , Arsenic trioxide , Sodium selenite
- 주제(KDC) 570
- 주제(DDC) 660
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2007
- 학위수여년월 2007. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Malignant gliomas, the most common brain tumors with high mortality, remain largely incurable despite multimodal treatments including surgical resection, radiotherapy, and chemotherapy. Thus, the researchers are currently attempting to develop novel therapeutic strategies for malignant gliomas. Here, we show that treatment with subtoxic doses of HDAC inhibitor (sodium butyrate, tricostatin A or SBHA) or As2O3 in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, but not in human astrocytes. HDAC inhibitor sensitizes human glioma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and the subsequent downregulation of surviving and XIAP, whereas As2O3 sensitizes human glioma cells to TRAIL-induced apoptosis via CHOP-dependent DR5 upregulation. Furthermore, we demonstrate that selenite is preferentially cytotoxic to various human glioma cells over normal astrocytes via autophagic cell death. In this process, selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death. Therefore, these results suggest that stimulation of TRAIL-induced apoptosis by co-treated HDAC inhibitor or arsenic trioxide as well as selenite-induced autophagic cell death may offer an attractive strategy for safely treating gliomas.
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TABLE OF CONTENTS
ABSTRACT - i
TABLE OF CONTENTS -iii
LIST OF FIGURES -viii
LIST OF TABLES -xiv
I. INTRODUCTION -1
A. Malignant glioma -1
B. Programmed cell death -2
1. Apoptosis - 4
2. Autophagic cell death -8
3. Necrosis -11
C. TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) -12
D. IAPs (Inhibitor of apoptosis proteins) -14
II. MATERIALS AND METHODS -22
A.Chemicals and antibodies - 22
B.Culture of glioma cell lines and normal human astrocytes - 23
C.Measurement of cellular viability - 24
D.Immunoblotting - 24
E.RT-PCR - 25
F.Cdc2 and Cdk2 Immune Complex Kinase Assay -26
G.Flow cytometry of death receptors - 27
H.Establishment of the stable cell lines overexpressing CrmA, Akt, Bcl-2, Bcl-xL, XIAP, survivin, Cu/ZnSOD, MnSOD or catalase and the stable cell lines expressing the fluorescence specifically in mitochondria, endoplasmic reticulum (ER) or peroxisomes - 27
I.Expression of Cdc2, Cdk2, cyclin A or cyclin B by transient transfection -28
J.Cyclin A and Cyclin B luciferase assay -29
K.DR5 and CHOP luciferase assay -30
L.Small interfering RNAs - 31
M.Detection and quantification of acidic vesicular organelles (AVOs) with acridine orange staining -31
N.Visualization of autophagic vacuoles - 32
O.GFP-LC3 translocation - 33
P.Measurement of mitochondrial membrane potential (MMP) - 32
Q.Transmission electron microscopy - 33
R.Measurement of superoxide anion - 33
S.Measurement of ROS - 33
T.Measurement of SOD and catalase activity - 33
U.Construction of retroviral vectors encoding Cu/ZnSOD, MnSOD or catalase and their transduction - 34
V.Statistical analysis - 35
- Chapter I
Sodium Butyrate Sensitizes Human Glioma Cells to TRAIL-Mediated Apoptosis Through Inhibition of Cdc2 and the Subsequent Downregulation of Survivin and XIAP - 36
ABSTRACT - 37
A.INTRODUCTION - 39
B.RESULTS - 41
1.Sodium butyrate sensitizes TRAIL-induced apoptosis in glioma cells but not in normal astrocytes - 41
2.Co-treatment with sodium butyrate and TRAIL recovers incomplete activation of caspases in TRAIL-resistant glioma cells - 46
3.Combined treatment with sodium butyrate and TRAIL reduces the expression of survivin and XIAP - 52
4.Sodium butyrate enhances TRAIL-induced apoptosis by downregulation of survivin and XIAP - 58
5.Sodium butyrate inhibits Cdc2 and Cdk2 activity through modulation of the expressions of various cell cycle regulators - 61
6.Forced expression of Cdc2 plus cyclin B attenuates sodium butyrate-stimulated TRAIL-induced apoptosis - 66
7. Other HDAC inhibitors also sensitize glioma cells to TRAIL-induced apoptosis through Cdc2-mediated downregulation of survivin and XIAP - 69
C.DISCUSSION - 76
- Chapter II
Arsenic Trioxide Sensitizes Human Glioma Cells, but not Normal Astrocytes, to TRAIL-Induced Apoptosis via CHOP-Dependent DR5 Upegulation - 84
ABSTRACT - 85
A.INTRODUCTION - 87
B.RESULTS - 89
1.As2O3 sensitizes human glioma cells to TRAIL-induced apoptosis - 89
2.Cotreatment with As2O3 and TRAIL recovers incomplete activation of caspases in TRAIL-resistant glioma cells - 91
3. Neither BNIP3 induction nor PARP activation is important for As2O3–stimulated TRAIL-mediated apoptosis - 95
4.DR5 upregulation plays a critical role in As2O3-induced sensitization of TRAIL-induced apoptosis of various glioma cells - 100
5.ROS generation is not involved in the enhanced apoptosis by the combined treatment with As2O3 and TRAIL - 106
6.As2O3 induced DR5-upregulation in glioma cells is CHOP-dependent - 111
7.Human astrocytes are resistant to As2O3–induced DR5 up-regulation and to TRAIL-induced partial proteolytic processing of procaspase-3 - 118
C. DISCUSSION - 123
-Chapter III
Sodium Selenite Induces Superoxide-Mediated Mitochondrial Damage and Subsequent Autophagic Cell Death in Malignant Glioma Cells - 131
ABSTRACT - 132
A.INTRODUCTION- 134
B.RESULTS - 136
1.Sodium selenite induces non-apoptotic cell death in human glioma cells - 136
2.Selenite induces autophagy in glioma cells -144
3.Selenite-damaged mitochondria are targeted by autophagy -153
4.Selenite-induced superoxide generation triggers mitochondrial damage and subsequent autophagic cell death - 162
C.DISCUSSION - 176
III. CONCLUSION - 181
REFERENCES - 184
국문요약 - 215
