간암 세포에서 doxorubicin 저항성 극복을 위한 전략 연구
Study on the Strategies to Overcome Doxorubicin Resistance in Hepatoma Cells
- 주제(키워드) Cdc2 , Cdk2 , doxorubicin , mitotic catastrophe , apoptosis , hepatoma , Bcl-xL , PDTC , autophagy
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2010
- 학위수여년월 2010. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010555
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Study on the strategies to overcome doxorubicin resistance in hepatoma cells In Huh-7 hepatoma cells, low dose (LD) doxorubicin treatment induces cell death through mitotic catastrophe accompanying the formation of large cells with multiple micronuclei, whereas high dose (HD) doxorubicin induces apoptosis. In part I, I investigated the role of Cdc2 and Cdk2 kinase in the regulation of the two modes of cell death induced by doxorubicin, apoptosis and cell death through mitotic catastrophe. During HD doxorubicin-induced apoptosis, the histone H1-associated activities of Cdc2 and Cdk2 both progressively declined in parallel with reductions in cyclin A and cyclin B protein levels. In contrast, during LD doxorubicin-induced cell death through mitotic catastrophe, the Cdc2 and Cdk2 kinases were transiently activated 1 day post-treatment, with similar changes seen in the protein levels of cyclin A, cyclin B and Cdc2. Treatment with roscovitine, a specific inhibitor of Cdc2 and Cdk2, significantly blocked LD doxorubicin-induced mitotic catastrophe and cell death, but did not affect HD doxorubicin-induced apoptosis in Huh-7, SNU-398 and SNU-449 hepatoma cell lines. Our results demonstrate that differential regulation of Cdc2 and Cdk2 activity by different doses of doxorubicin may contribute to the induction of two distinct modes of cell death in hepatoma cells, either apoptosis or cell death through mitotic catastrophe. Bcl-xL is often overexpressed in human hepatocellular carcinoma cells, contributing to resistance to various chemotherapeutic agents. I investigated the role of Bcl-xL in two modes of cell death induced by different doses of doxorubicin, apoptosis and cell death through mitotic catastrophe. Bcl-xL overexpression in various hepatoma cells effectively blocked apoptosis induced by high dose doxorubicin, inhibiting the loss of mitochondrial membrane potential, release of mitochondrial cytochrome c and caspase activation. Contrastingly, Bcl-xL overexpression did not block low dose doxorubicin-induced mitotic catastrophe and subsequent non-apoptotic cell death, without affecting abnormal cell cycle progression, formation of multiple micronuclei, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and the clonogenicity of cells exposed to low dose doxorubicin. These findings indicate that low dose doxorubicin-induced cell death through mitotic catastrophe may provide an alternative therapeutic strategy for Bcl-xL-overexpressing hepatoma cells, which are resistant to pro-apoptotic treatments. In part II, I investigated whether co-treated pyrrolidine dithiocarbamate (PDTC) might overcome Bcl-xL-mediated resistance to doxorubicin in hepatoma cells. I found that combined treatment with PDTC and doxorubicin induced the cell death accompanying autophagy in Bcl-xL-overexpressing Chang liver and hepatoma cells. Contrastingly, co-treated PDTC attenuated doxorubicin-induced apoptosis in Chang liver cells and hepatocytes. Thus, combined regimen with PDTC and doxorubicin may provide a safe and effective therapeutic strategy for malignant hepatoma with Bcl-xL-mediated apoptotic defects.
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TABLE OF CONTENTS
ABSTRACT---------------------------------------------------------------------------------------------- i
TABLE OF CONTENTS------------------------------------------------------------------------------ iv
LIST OF FIGURES------------------------------------------------------------------------------------ vii
I. INTRODUCTION----------------------------------------------------------------------------- 1
II. MATERIALS AND METHODS-------------------------------------------------------------- 7
A. Reagents-------------------------------------------------------------------------------------- 7
B. Induction of cell death through mitotic catastrophe or apoptosis by different doses of doxorubicin-------------------------------------------------------------------------------- 7
C. Staining of nuclei----------------------------------------------------------------------------- 8
D. Measurement of cellular viability---------------------------------------------------------- 8
E. DNA fragmentation analysis by agarose gel electrophoresis-------------------------- 9
F. TUNEL assay---------------------------------------------------------------------------------9
G. Cell cycle analysis--------------------------------------------------------------------------- 9
H. Cdc2 and Cdk2 immune complex kinase assay----------------------------------------10
I. Establishment of the stable hepatoma cell lines overexpressing Bcl-xL-------------11
J. Clonogenic assay-----------------------------------------------------------------------------11
K. Immunocytochemistry-----------------------------------------------------------------------11
L. Measurement of mitochondrial membrane potential (MMP)--------------------------12
M. Isolation of cytosolic fractions for analysis of the release of mitochondrial proteins
----------------------------------------------------------------------------------------------- 12
N. Measurement of ROS------------------------------------------------------------------------12
O. Detection of LC3 translocation into autophagic vacuoles------------------------------13
P. Detection of MDC uptake into autophagic vacuoles------------------------------------13
Q. Transmission electron microscopy--------------------------------------------------------13
R. Glutathione assay----------------------------------------------------------------------------14
S. Statistical Analysis---------------------------------------------------------------------------14
III. RESULTS-------------------------------------------------------------------------------------15
Part I
A. Different does of doxorubicin induces two distinct modes of cell death, cell death through mitotic catastrophe and apoptosis----------------------------------------------- 15
B. Expression of cell cycle regulators are differently regulated during the
progression of doxorubicin-induced cell death through mitotic catastrophe
and apoptosis---------------------------------------------------------------------------------22
C. Cdc2 and Cdk2 play critical role in LD doxorubicin-induced cell death through mitotic catastrophe but not in HD doxorubicin-induced apoptosis
------------------------------------------------------------------------------------------------26
D. Inhibition of Cdc2 and Cdk2 activity blocks LD doxorubicin-induced mitotic catastrophe------------------------------------------------------------------------------------ 29
E. Overexpression of Bcl-xL in Huh-7 cells effectively blocks high dose doxorubicin-induced apoptosis but not LD doxorubicin-induced cell death through mitotic catastrophe------------------------------------------------------------------------------------ 35
F. The cell cycle is dysregulated in control and Bcl-xL-overexpressing Huh-7 cells treated with LD doxorubicin----------------------------------------------------------------47
G. LD doxorubicin-induced loss of MMP and release of mitochondrial cytochrome c are not blocked by Bcl-xL overexpression------------------------------------------------51
H. LD doxorubicin-induced cell death through mitotic catastrophe also occurs in other Bcl-xL-overexpressing hepatoma cells--------------------------------------------------- 56
Part II
I. Dxorubicin-induced apoptosis in Chang normal liver cells is attenuated by PDTC-----------------------------------------------------------------------------------------------------61
J. Bcl-xL-mediated resistance to doxorubicin ca be recovered by co-treated PDTC-66
K. Combined treatment with PDTC and doxorubicin induces autophagy in Bcl-xL-overexpressing Chang cells-----------------------------------------------------------------71
L. Cell death accompanying autophagy is also induced by the combined
treatment with PDTC plus doxorubicin in other hepatoma cells
overexpressing Bcl-xL-----------------------------------------------------------------------75
M. Antioxidant activity of PDTC may contribute to its cytoprotective effect on doxorubicin-induced apoptosis-------------------------------------------------------------78
N. Other mechanism by PDTC plus doxorubicin in Bcl-xL-overexpressing cells may contribute to induction of cell death accompanying autophagy------------------------82
IV. DISCUSSION-----------------------------------------------------------------------------------85
V. CONCLUSION---------------------------------------------------------------------------------102
REFERENCES ----------------------------------------------------------------------------------------104
국문요약-----------------------------------------------------------------------------------------------117
