TRAIL에 저항성을 가진 악성 암세포에 quercetin 또는 capsaicin과 TRAIL의 병합 처리 시 세포사멸 촉진 효과 및 조절 기전 분석
Sensitization of TRAIL-mediated apoptosis by quercetin or capsaicin in malignant cancer cells
초록/요약
Quercetin (3’,3’,4’,5,7-pentahydroxyflavone), a flavonoid found in many fruits and vegetables, is known to have anti-cancer effects. Here, we show that treatment with subtoxic doses of quercetin in combination with tumor necrosis factor?related apoptosis-inducing ligand (TRAIL) significantly induce rapid apoptosis in TRAIL?resistant hepatocellular carcinoma (HCC) cells. Effective induction of apoptosis by the combined treatment with quercetin and TRAIL was not blocked by overexpression of Bcl-xL, which is known to be highly expressed in HCC cells conferring resistance to various chemotherapeutic agents, suggesting that this combined treatment may provide an attractive strategy for treating resistant HCCs. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in various HCC cells, treatment with quercetin efficiently recovered TRAIL-induced activation of caspases. We found that quercetin treatment significantly up?regulated mRNA and protein levels of DR5, a death receptor of TRAIL. Furthermore, treatment with quercetin significantly decreased the protein levels of c-FLIP, a dominant negative inhibitor of caspase-8. Either administration of small interfering RNA for DR5 or overexpression of c-FLIPs, but not c-FLIPL, significantly attenuated quercetin-stimulated TRAIL-induced apoptosis, demonstrating their contribution to overcoming TRAIL-resistance of HCC cells. Collectively, quercetin sensitizes TRAIL-resistant HCC cells to TRAIL-induced apoptosis via up-regulation of DR5 and downregulation of c-FLIPs.
more초록/요약
Tumor Necrosis Factor(TNF) family 인 Tumor Necrosis Factor-related Apoptosis Inducing Ligand(TRAIL)은 정상 세포에는 거의 영향을 미치지 않으면서, 다양한 암세포에서 효과적으로 apoptosis를 일으킴으로써, 새로운 항암 치료책으로의 가능성을 보여 주었다. 그러나 최근 들어 TRAIL에 의한 apoptosis 감수성이 암세포에 따라 다르게 나타나며, TRAIL receptor 들을 발현하는 많은 암세포들 또한 TRAIL에 resistant한 것으로 보고 되고 있다. 따라서 본 연구에서는 암세포에서 TRAIL 저항성을 극복할 수 있는 효과적인 방법으로 subtoxic한 농도의 quercetin 또는 capsaicin과 TRAIL을 병합 처리할 경우, TRAIL 저항성을 가진 악성 암세포에서 급격한 apoptosis를 유도하는 것을 관찰 하였다. 또한 이 두 약제는 TRAIL을 단독 처리할 경우 악성 암세포에서 procaspase-3의 proteolytic processing이 일부 단계에서 차단되는 반면, 두 약제들과 병합처리할 경우 차단되었던 proteolytic processing step이 풀리면서 TRAIL에 의한 caspase 활성화가 복구, 회복 됨을 확인 하였다. Quercetin의 처리 시에 TRAIL death receptor인 DR5의 발현 양 증가를 발견하여, DR5를 target으로 하는 small interfering RNAs를 처리할 경우 quercetin에 의한 TRAIL sensitiziation을 효과적으로 차단됨을 확인할 수 있었다. 이와 더불어, quercetin을 처리할 경우 c-FLIP의 단백질 발현양이 현저하게 감소되며, 과 발현 시킬 경우 quercetin을 매개로 한 TRAIL 유도 apoptosis가 차단 됨을 알 수 있었다. Capsaicin의 처리 시에 survivin의 단백질 발현 양이 현저하게 감소되며, 이 또한 과 발현 시킬 경우 capsaicin을 매개로 한 TRAIL 유도 apoptosis 가 차단 됨을 알 수 있었다. 이를 통해 본 결과는 quercetin 처리로 인한 DR5의 upregulation과 c-FLIP의 downregulation이, capsaicin 처리로 인한 survivin의 downregulation이 악성 암세포에서 TRAIL 저항성을 극복하는데 기여 한다는 결론을 도출 할 수 있었으며 이 두 약제는 악성 암세포에 좋은 치료 전략이 될 수 있을 것이다.
more목차
Chapter ⅠQuercetin sensitizes human hepatoma cells to TRAIL?induced apoptosis by Sp1-mediated up-regulation of DR5 and proteasome-mediated down-regulation of c-FLIP_(S) = ⅰ
ABSTRACT = ⅱ
TABLE OF CONTENTS = ⅳ
LIST OF FUGURES = ⅶ
Ⅰ. INTRODUCTION = 1
Ⅱ. MATERIALS AND METHODS = 4
A. Chemicals and antibodies = 4
B. Culture of hepatoma and glioma cells = 5
C. Measurement of cellular viability = 5
D. Immunoblotting = 5
E. DNA fragmentation assay = 6
F. Reverse transcription-PCR analysis = 7
G. Flow cytometry of death receptors = 8
H. Small interfering RNA = 8
I. Plasmids = 9
J. Transfections and luciferase assays = 10
K. Construction of the expression vectors for c-FLIP_(L) and c-FLIP_(S) = 11
L. Establishment of the cell lines stably overexpressing Bcl-2, Bcl-xL, FLIP_(L) or FLIP_(S) = 11
Ⅲ. RESULTS = 13
1. Subtoxic doses of quercetin significantly sensitize TRAIL-resistant hepatoma cells to TRAIL-induced apoptosis = 13
2. TRAIL-induced incomplete proteolytic processing of capase-3 in hepatoma cells is completed by co-treated quercetin = 17
3. Neither Akt inhibition nor survivin downregulation is a common mechanism for quercetin-induced TRAIL-mediated apoptosis in hepatoma cells = 24
4. Quercetin up-regulates DR5 in various hepatoma cells = 30
5. Sp1 mediates quercetin-induced DR5 up-regulation = 36
6. Proteasome-mediated degradation of c-FLIPS may be critical for quercetin-sensitized TRAIL-induced apoptosis = 47
Ⅳ. DISCUSSION = 55
Chapter Ⅱ Capsaicin sensitizes TRAIL-mediated apoptosis through survivin downregulation = 64
ABSTRACT = 65
Ⅰ. INTRODUCTION = 66
Ⅱ. RESULTS = 68
1. Subtoxic doses of capsaicin significantly enhanced cell death in TRAIL-resistant human glioma cell lines = 68
2. Combined treatment with capsaicin and TRAIL enhanced cell death in glioma cells overexpressing Bcl-2 or Bcl-xL but not in human normal astrocytes = 72
3. Combine treatment with Capsaicin and TRAIL induced apoptotic cell death = 75
4. Capsaicin sensitizes TRAIL-induced apoptosis in TRPV1 independent pathway = 79
5. Capsaicin enhances TRAIL-induced apoptosis by downregulation of survivin = 81
Ⅲ. DISCUSSION = 88
CONCLUSION = 91
REFERENCES = 92
국문요약 = 110
