Silibinin과 TRAIL 병합처리에 의한 glioma 세포 사멸 유도 기전 분석
Regulatory mechanisms involved in the cell death of human glioma cells treated with silibinin and TRAIL
초록/요약
Silibinin은 milk thistle에서 추출한 flavonoid물질로서 Chemopreventive agent로 주목을 받고 있다. 본 연구에서는, subtoxic한 농도의 silibinin 과 TRAIL (tumor necrosis factor-related apoptosis-inducding ligand)을 병합 처리할 경우, TRAIL 저항성을 가진 뇌암 세포에 급격한 apoptosis를 유도할 수 있는 반면, 정상 뇌세포인 astrocytes에는 아무런 영향을 주지 않았다. TRAIL을 단독 처리할 경우 뇌암 세포에서 procaspase-3의 proteolytic processing이 일부 단계에서 차단되는 반면, silibinin을 병합 처리할 경우 차단되었던 proteolytic processing step이 풀리면서 TRAIL에 의한 caspase 활성화가 복구, 회복 됨을 확인하였다. 또한 slilibinin의 처리를 통해 transcription factor인 CHOP/GADD153의 단백질 발현양의 증가와 TRAIL death receptor인 DR5의 발현양 증가를 발견하여, CHOP과 DR5를 target으로 하는 small interfering RNAs 를 처리할 경우 silibinin에 의한 TRAIL sensitization 을 효과적으로 차단됨을 확인할 수 있었다. 이와 더불어, silibinin을 처리할 경우 survivin과 c-FLIP 의 단백질 발현양이 현저하게 감소되며, 이들을 과 발현 시킬 경우, silibinin을 매개로 한 TRAIL 유도 apoptosis가 차단 됨을 알 수 있었다. 이를 통해 본 결과는 subtoxic한 농도의 silibinin 처리로 인해, CHOP을 통한 DR5의 upregulation과 survivin, c-FLIP의 downregulation이 뇌암 세포에서 TRAIL저항성을 극복하는데 기여 한다는 결론을 도출 할 수 있었다.
more초록/요약
Silibinin, a flavonoid isolated from milk thistle, has been reported to have cancer chemopreventive and therapeutic efficacy. Here we show that treatment with subtoxic doses of silibinin in combination with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, but not in human astrocytes, suggesting that this combined treatment may offer an attractive strategy for safely treating gliomas. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in glioma cells, treatment with silibinin efficiently recovered TRAIL-induced activation of caspases. We found that silibinin treatment up-regulated DR5, a death receptor of TRAIL, in a transcription factor CHOP-dependent manner. Furthermore, treatment with silibinin downregulated the protein levels of FLIPL, FLIPs, and survivin through proteasome-mediated degradation. Taken together, our results demonstrate that the ability of silibinin to modulate multiple components in the death receptor-mediated apoptotic pathway is responsible for the effective recovery of TRAIL sensitivity in TRAIL-resistant glioma cells.
more목차
Ⅰ. Introduction = 1
Ⅱ. Materials and Methods = 3
A. Reagents = 3
B. Cells and Culture Conditions = 3
C. Measurement of Cellular Viability = 4
D. Construction of the expression vectors for c-FLIPL and c-FLIPS = 4
E. Establishment of the stable cell lines overexpressing CrmA, Akt, survivin, c-FLIPL or c-FLIPs = 4
F. RT-PCR = 5
G. Flow cytometry of death receptors = 6
Small interfering RNAs = 6
Ⅲ. RESULTS = 8
1. Subtoxic doses of silibinin effectively sensitize TRAIL-mediated apoptosis in TRAIL-resistant glioma cells but not in normal astrocytes = 8
2. Critical role of caspases in cell death by the combined treatment with silibinin and TRAIL = 10
3. CHOP-mediated DR5 upregulation contributes to silibinin-stimulated TRAIL-induced apoptosis = 15
4. Downregulaiton of FLIP and survivin is also involved in silibinin-facilitated TRAIL-induced apoptosis = 24
DISCUSSION = 40
Ⅴ. CONCLUSION = 45
References = 46
국문요약 = 56
