Role of Bcl-xL in Doxorubicin-Induced Cell Death: Apoptosis and Mitotic Cell Death
- 발행기관 The Graduate School, Ajou University
- 지도교수 Choi, Kyeong Sook
- 발행년도 2004
- 학위수여년월 2005. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의학계열
- 본문언어 영어
초록/요약
Bcl-xL overexpressed in various human cancer cells contributes to resistance against various chemotherapeutic agents. In this study, we compared the role of Bcl-xL in high dose (HD) doxorubicin-induced apoptosis and low dose (LD) doxorubicin-induced mitotic cell death. In the first part of this study, we investigated whether anti-apoptotic effect of Bcl-xL on doxorubicin-induced apoptosis was associated with Bcl-xL-mediated modulation of signaling pathways. Bcl-xL overexpression in many cell types effectively blocked HD doxorubicin-induced apoptosis. HD Doxorubicin-induced activation of p38 or JNK was not affected by Bcl-xL overexpression. However, ERK2 was rapidly activated and its high activity was sustained in Bcl-xL-overexpressing cells treated with doxorubicin, whereas its activity was progressively decreased in doxorubicin-treated control cells. CREB, p90RSK, and NF-B, possible target molecules of ERK2, were also activated in Bcl-xL-overexpressing cells treated with doxorubicin. Enhanced expression of wild type ERK2 in control cells alleviated doxorubicin-induced apoptosis. Moreover, forced expression of dominant-negative ERK2 mutant in Bcl-xL overexpressing cells significantly increased the sensitivity to doxorubicin-induced apoptosis, suggesting the critical role of ERK2 in Bcl-xL-mediated blocking of doxorubicin-induced apoptosis. ERK2 activation was not observed in Bcl-2-overexpressing cells treated with doxorubicin, although Bcl-2 overexpression also could inhibit doxorubicin-induced apoptosis. These results suggest that doxorubicin-induced ERK2 activation is specifically mediated by Bcl-xL. Therefore, our results demonstrate that Bcl-xL-mediated ERK2 activation may provide one mechanism by which BcL-xL confers caner cells resistance to doxorubicin-induced apoptosis. In the second part of this study, we investigated whether Bcl-xL overexpression affected senescence and/or mitotic cell death induced by LD of doxorubicin. Bcl-xL overexpression effectively blocked apoptosis induced by high dose HD of doxorubicin, blocking the loss of mitochondrial membrane potential and activation of caspases in Huh-7 cells. In contrast, overexpression of Bcl-xL slightly delayed but not completely blocked doxorubicin-induced senescence-like phenotype (SLP) and the subsequent mitotic cell death. Loss of mitochondrial function and downregulation of mitosis-controlling proteins were also slightly delayed but not abolished by Bcl-xL overexpression. Clonogenicity of hepatoma cells exposed to LD doxorubicin was not enhanced by Bcl-xL overexpression. These results suggest that induction of senescence and/or mitotic cell death by LD doxorubicin may be more effective for Bcl-xL-overexpressing hepatoma cells, which are resistant to the apoptotic effect of chemotherapeutic agents.
more초록/요약
Bcl-xL은 많은 종류의 암에 과발현되며 다양한 항암제에 의한 mitochondria의 기능 감소를 막음으로써 항암제 내성을 유발한다. Doxorubicin은 현재 가장 많이 쓰이고 있는 항암제 중 하나이지만, 그 작용 기전이 아직 명확히 규명되지 않고 있다. 본 연구에서는 고농도 doxorubicin에 의해 유도되는 apoptosis와 저농도 doxorubicin에 의해 유도되는 mitotic cell death에서의 Bcl-xL의 기능에 관해 살펴보았다. Bcl-xL이 apoptosis를 막는다는 것은 잘 알려져 있으나, Bcl-xL이 항암제에 반응하여 다른 신호전달체계의 조절을 통해 apoptosis를 막을 수 있음을 보고한 예는 아직까지 없다. 본 연구에서는 Bcl-xL 과발현 세포에 고농도의 doxorubicin을 처리했을 경우 apoptosis가 현저하게 막아졌으며, 이때 MAP kinase 중 ERK2만 활성화되었고 그 downstream 불질인 p90RSK, CREB, NF-B가 활성화되는 것을 확인하였다. Bcl-xL 과발현 세포에서 doxorubicin에 반응하여 활성화되는 ERK2는 doxorubicin에 대한 저항성을 가지게 하는데 중요한 역할을 함을 알 수 있었다. Bcl-2 과발현 또한 doxorubicin에 의한 apoptosis를 막을 수 있으나 ERK2는 활성시키지 못하였다. 다음으로, Bcl-xL의 과발현이 mitotic cell death에 미치는 영향에 대해 조사해보았다. Mitotic cell death 시에는 mitochondria의 기능이 어떻게 변화하는지, 또한 Bcl-xL이 어떤 역할을 하는지 전혀 밝혀져 있지 않은 실정이다. 본 연구를 통해 Bcl-xL 과발현이 저농도 doxorubicin에 의해 유도되는 mitotic cell death를 막지 못한다는 것을 알 수 있었다. Bcl-xL을 과발현시킬 경우 apoptosis 시와는 달리 mitotic cell death 시에 수반되는 mitochondria 기능 감소를 억제하지 못하였다. 따라서, Bcl-xL 과발현으로 인해 apoptosis에 내성을 획득하게 된 암의 치료에 있어서, 저농도 doxorubicin을 이용해 mitotic cell death를 유도하는 것이 효과적인 대안이 될 수 있을 것으로 본다.
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Contents
ABSTRACT = i
TABLE OF CONTENTS = ⅱ
LIST OF FIGURES = ⅴ
LIST OF TABLE = ⅸ
Ⅰ. INTRODUCTION = 1
Ⅱ. MATERIALS AND METHODS = 5
A. Chemicals and antibiotics = 5
B. Cell culture = 5
C. Measurement of cellular viability = 6
D. Bcl-xL-, Bcl-2-, XIAP-, Akt- or survivin overexpressing stable cell lines = 6
E. Immunoblotting = 6
F. Immunocytochemistry = 7
G. SA-β-gal activity = 8
H. Detection of mitochondrial permeability transition = 8
I. Detection of ATP depletion = 9
J. Subcellular fractionation = 9
K. Cell cycle analysis = 10
L. Clonogenic assay = 10
M. Expression of dominant-negative ERK2 or wild type ERK2 by transient transfection = 11
Ⅲ. RESULTS = 12
A. Bcl-xL-mediated ERK2 activation plays a critical role in the inhibition of doxorubicin-induced apoptosis = 12
1. Bcl-xL overexpression blocks doxorubicin-induced apoptosis in Huh-7 hepatoma cells = 12
2. ERK2 is activated in Bcl-xL overexpressing Huh-7 cells treated with HD doxorubicin = 12
3. Overexpression of Bcl-xL in other cell lines also blocks doxorubicin-induced apoptosis accompanying the activation of ERK2 = 13
4. ERK2 activation is important for the blocking effect of Bcl-xL on doxorubicin-induced apoptosis = 20
5. Activation of ERK2 can inhibit the release of mitochondrial cytochrome c = 21
6. Doxorubicin-induced ERK2 activation is specifically mediated by Bcl-xL = 26
B. Bcl-xL blocks apoptosis but not mitotic cell death induced by doxorubicin = 29
1. Bcl-xL overexpression blocks doxorubicin-induced apoptosis in Huh-7 hepatoma cells = 29
2. Bcl-xL delays LD doxorubicin-induced mitotic cell death = 29
3. Bcl-xL does not inhibit LD doxorubicin-induced aberrant cell cycle progression and downregulation of cell cycle regulators = 30
4. Missegregation of chromosomes and loss in the integrity of nuclear membranes are observed in both Huh-7 and Bcl-xL overexpressing Huh-7 cells treated with LD doxorubicin = 31
5. Bcl-xL overexpression does not inhibit loss of mitochondrial function and release of inner-mitochondrial proteins into cytosol in LD doxorubicin-induced mitotic cell death = 39
6. Not only Bcl-xL but also Bcl-2 does not block mitotic cell death = 47
7. Bcl-xL does not enhance long-survival of the cells treated with LD doxorubicin = 47
Ⅳ. DISCUSSION = 52
A. Bcl-xL-mediated ERK2 activation plays a critical role in the inhibition of doxorubicin-induced apoptosis = 52
B. Bcl-xL blocks apoptosis but not mitotic cell death induced by doxorubicin = 58
Ⅴ. CONCLUSION = 61
REFERENCES = 62
국문요약 = 72
목차
Fig. 01. Chemosensitivity of Huh-7, U87MG and Chang cells toward doxorubicin treatment = 15
Fig. 02. Bcl-xL blocks doxorubicin-induced apoptosis = 17
Fig. 03. ERK2 is activated in Bcl-xL overexpressing cells treated with doxorubicin = 18
Fig. 04. Bcl-xL overexpression in Chang or U87MG cells blocks doxorubicin-induced apoptosis accompanying the activation of ERK2 = 19
Fig. 05. Forced expression of DN ERK2 enhances doxorubicin-induced apoptosis in Bcl-xL overexpressing cells = 22
Fig. 06. Forced expression of WT ERK2 attenuated doxorubicin-induced apoptosis in Chang cells = 23
Fig. 07. Effect of WT ERK2 overexpression in control cells or DN ERK2 overexpression in Bcl-xL-overexpressing cells on doxorubicin-induced release of mitochondrial cytochrome c = 24
Fig. 08. Bcl-2 overexpression in U87MG cells blocks doxorubicin-induced apoptosis without accompanying activation of ERK2 = 26
Fig. 09. ERK2 is not activated in Bcl-2-overexpressing Huh-7 cells treated with doxorubicin = 27
Fig. 10. Bcl-xL did not block LD doxorubicin-induced multinucleation = 32
Fig. 11. Bcl-xL delays LD doxorubicin-induced senescence = 33
Fig. 12. Bcl-xL delays but not block LD doxorubicin-induced mitotic cell death in Huh-7 cells = 34
Fig. 13. Bcl-xL does not inhibit aberrant cell cycle progression and downregulation of cell cycle regulators = 35
Fig. 14. Missegregation of chromosomes is observed not only in Huh-7 cells but also in Bcl-xL-overexpressing Huh-7 cells treated with LD doxorubicin = 36
Fig. 15. Loss in the integrity of nuclear membrane is observed in both Huh-7 and Bcl-xL-overexpressing Huh-7 cells treated with LD doxorubicin = 37
Fig. 16. Bcl-xL does not block LD doxorubicin-induced loss of mitochondrial membrane potential = 40
Fig. 17. Bcl-xL slightly delays doxorubicin-induced loss of intracellular ATP levels = 41
Fig. 18. Release of cytochrome c is observed not only in Huh-7 cells but also in Bcl-xL overexpressing cells treated with LD doxorubicin = 42
Fig. 19. Release of Smac is observed not only in Huh-7 cells but also in Bcl-xL overexpressing cells treated with LD doxorubicin = 43
Fig. 20. Bcl-xL blocks HD doxorubicin-induced loss of mitochondrial membrane potential and ATP levels = 44
Fig. 21. Bcl-xL blocks HD doxorubicin-induced release of mitochondrial cytochrome c and Smac into cytosol = 45
Fig. 22. Bcl-xL overexpression in Chang cells blocks HD doxorubicin-induced apoptosis but not LD doxorubicin-induced mitotic cell death = 48
Fig. 23. Bcl-2 does not block LD doxorubicin-induced mitotic cell death = 49
Fig. 24. Bcl-xL does not enhance long-term survival of the cells treated with LD doxorubicin = 50
Fig. 25. Schematic model for Bcl-xL mediated ERK2 activation in response to doxorubicin = 56
목차
Table. 01. Determination of the cytotoxic effects of doxorubicin on Huh-7, U87MG and Chang cells = 16
