TRAIL에 저항성을 가진 간암세포에 Sulforaphan과 TRAIL 의 병합 처리 시 apoptosis촉진 효과 및 조절 기전 분석 : Sulforaphane 에 의한 반응성 산소종 생성과 DR5 발현 유도의 중요성 분석
Sulforaphane sensitizes TRAIL-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen species-mediated upregulation of DR5
초록/요약
Sulforaphane (SFN) is a chemopreventive agent present in various cruciferous vegetables, including broccoli. Here, we show that treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with subtoxic doses of SFN significantly induces rapid apoptosis in TRAIL-resistant hepatoma cells. Neither TNF-α- nor Fas-mediated apoptosis was sensitized in hepatoma cells by cotreatment with SFN, suggesting that SFN can selectively sensitize cells to TRAIL-induced apoptosis but not to apoptosis mediated by other death receptors. We found that SFN treatment significantly upregulated mRNA and protein levels of DR5, a death receptor of TRAIL. This was accompanied by an increase in the generation of reactive oxygen species. Pretreatment with N-acetylcysteine and overexpression of catalase inhibited SFN-induced upregulation of DR5 and almost completely blocked the cotreatment-induced apoptosis. Furthermore, the SFN-mediated sensitization to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs for DR5. These results collectively indicate that SFN-induced generation of reactive oxygen species and the subsequent upregulation of DR5 are critical for triggering and amplifying TRAIL-induced apoptotic signaling. We also found that SFN can sensitize both Bcl-xL- and Bcl-2-overexpressing hepatoma cells to TRAIL-induced apoptosis, indicating that treatment with a combination of TRAIL and SFN may be a safe strategy for treating resistant hepatomas.
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TABLE OF CONTENTS
ABSTRAT ⅰ
TABLE OF CONTENTS ⅲ
LIST OF FIGURES ⅴ
Ⅰ. INTROUDUCTION 1
Ⅱ. MATERIALS AND METHODS 3
A.Reagents 3
B.Cell cultures 3
C.Measurement of cellular viability 4
D.FACS analysis 4
E.DNA fragmentation assay 4
F.Immunoblotting 4
G.Plasmids, transfections and Luciferase assays 5
H.Semiquantitative RT-PCR analysis 6
I.Flow cytometry of death receptors 6
J.Small interfering RNA (siRNA) 7
K.Measurement of ROS 7
L.Construction of the expression vector encoding human Catalase 7
M.Establishment of the cell lines stably overexpressing Bcl-2, Bcl-xL or catalase 8
Ⅲ. RESULTS 9
1.Subtoxic doses of SFN significantly sensitize TRAIL-resistant hepatoma cells to TRAIL-induced apoptosis 9
2.SFN dose not affect the expression levels of IAPs or anti-apoptosis Bcl-2 family proteins 14
3.SFN upregulates DR5 in various hepatoma cells 18
4.SFN activates transcription from the DR5 promoter 25
5.SFN-induced ROS generation plays a critical role in the upregulation of DR5 and the induction of cell death by cotreatmnet with SFN and TRAIL 30
6.Combined treatment with SFN and TRAIL enhances cell death in hepatoma cells overexpressing Bcl-xL or Bcl-2 but not in rat primary Hepatocytes 37
Ⅳ.DISCUSSION 42
Ⅴ.CONCLUSION 47
REFERENCES 48
국문요약 55
