악성 뇌종양세포에서 paxilline에 의한 TRAIL 매개 apoptosis의 감작
Paxilline sensitizes malignant glioma cells to TRAIL-induced apoptosis via DR5 up-regulation and down-regulation of c-FLIP and survivin
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011018
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Although tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells, many cancer cells are resistant to TRAIL-induced cell death. We show here that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. Paxilline treatment markedly up-regulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly down-regulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5, or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
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ABSTRACT -i
TABLE OF CONTENTS -iii
LIST OF FIGURES -v
I. INTRODUCTION - 1
II. MATERIALS AND METHODS - 3
A. Chemicals and antibodies - 3
B. Culture of glioma cell lines and normal human astrocytes - 4
C. Measurement of Cellular Viability - 4
D. Reverse Transcription-PCR Analysis - 4
E. Small interfering RNA - 6
F. Plasmids, transfection, and luciferase assay - 6
G. Expression of c-FLIPS, c-FLIPL or survivin by transient transfection - 7
H. Establishment of the stable cell lines overexpressing c-FLIPS, c-FLIPL, or survivin - 8
I. Statistical Analysis and determination of synergy - 8
III. RESULTS - 10
1. Paxilline sensitizes malignant glioma cells to TRAIL-mediated cell death - 10
2. Paxilline recovers TRAIL sensitivity in glioma cells via induction of caspase-mediated apoptosis - 16
3. CHOP-dependent DR5 up-regulation plays an important role in the enhancement of TRAIL-induced apoptosis by paxilline - 26
4. Down-regulation of c-FLIP, in particular c-FLIPs, contributes to paxilline-triggered sensitization of glioma cells to TRAIL-induced apoptosis - 36
5. Survivin down-regulation also contributes to paxilline-induced sensitization of glioma cells to TRAIL-induced apoptosis - 44
6. Combined treatment with paxilline and TRAIL does not induce cell death in normal astrocytes without affecting CHOP, DR5, and survivin protein levels - 52
7. Inhibition of SERCA pump may contribute to the sensitizing effect of paxilline on TRAIL-mediated apoptosis - 58
IV. DISCUSSION - 63
V. REFERENCES - 71
VI. 국문요약 - 77
