악성 유방암 세포에서 paraptosis 유도 기전 연구
Study on the Induction of Paraptosis in Malignant Breast Cancer Cells
- 주제(키워드) Malignant breast cancer cells , Paraptosis , Mitochondrial Ca2+ , Superoxide , Proteasome inhibition
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2012
- 학위수여년월 2012. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000012820
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Defects in apoptotic signaling pathways contribute to the development of cancer and to therapy resistance (e.g. chemo- and radiotherapy) in many types of malignant tumors. Therefore, there is an urgent need to elucidate the molecular basis of apoptosis resistance and to develop novel strategies to overcome this resistance. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In this study, we investigated the role of mitochondrial superoxide and Ca2+ in curcumin-induced paraptosis. Curcumin induced mitochondrial Ca2+ overload and increased superoxide levels selectively in the malignant breast cancer cells, but not in the normal breast cell, contributing to the dilation of mitochondria/ER and subsequent paraptotic cell death. Combined treatment with the mitochondrial Na+/ Ca2+ exchanger inhibitor (mNCX; CGP-37157 or diltiazem) and a proteasome inhibitor (bortezomib, MG132 or lactacystin) effectively induced paraptotic cell death in MDA-MB 435S cells, whereas treatment with either agent alone did not. Thus, for effective induction of paraptosis in malignant breast cancer cells, simultaneous mitochondrial Ca2+ overload, mitochondrial superoxide generation and proteasomal inhibition are required. However, although curcumin exhibits growth-suppressive activity against a variety of cancer cells, its poor absorption and low systemic bioavailability of curcumin limit its translation into clinics as an anti-cancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated analogue of curcumin with improved stability, is significantly more potent than curcumin both in vitro and in vivo, while both DMC and curcumin were not cytotoxic to normal breast cells. DMC induced more effectively induced paraptosis, the cell death mode which is accompanied by dilation of mitochondria and the ER, than curcumin via stronger inhibition of proteasome and higher induction of CHOP. These results indicate that DMC exerts more potent anti-cancer effect on malignant breast cancer cells than curcumin through induction of paraptosis by stronger inhibition of proteasome activity.
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TABLE OF CONTENTS
ABSTRACT --------------------------------------------------------------------------------------- i
TABLE OF CONTENTS ---------------------------------------------------------------------- iii
LIST OF FIGURES ---------------------------------------------------------------------------- ix
LIST OF TABLES ----------------------------------------------------------------------------- xvi
I. INTRODUCTION ------------------------------------------------------------- 1
1. Malignant cancer cells and cell death ------------------------------------------------------ 1
1.1. Malignant cancer cells ------------------------------------------------------------------- 1
1.2. Different types of cell death in malignant cancer ------------------------------------- 5
1.3 Functional classification of regulated cell death modes ---------------------------- 10
A. Apoptosis --------------------------------------------------------------------------------- 10
(a) Extrinsic apoptosis -------------------------------------------------------------------- 10
(b) Intrinsic apoptosis --------------------------------------------------------------------- 12
B. Autophagic cell death ------------------------------------------------------------------- 14
C. Regulated necrosis: necroptosis ------------------------------------------------------- 17
D. Mitotic catastrophe ---------------------------------------------------------------------- 20
E. Alternative form of cell death ---------------------------------------------------------- 23
(a) Paraptosis ------------------------------------------------------------------------------- 23
(b) Parthanatos ----------------------------------------------------------------------------- 25
(c) Entosis ---------------------------------------------------------------------------------- 27
2. Promising anti-cancer drugs -------------------------------------------------------------- 30
2.1. Curcumin: Problems and Promises --------------------------------------------------- 30
A. Problems of curcumin bioavailability ------------------------------------------------ 31
B. Curcumin therapeutic Promises ------------------------------------------------------- 32
2.2. Michael acceptor ------------------------------------------------------------------------ 34
2.3. The cellular organelles as a chemotherapeutic target ------------------------------- 36
II. Materials and Methods -------------------------------------------------------------- 41
1. Chemicals and antibodies ------------------------------------------------------------------ 41
2. Cell culture of various cancer cell lines and normal cells ----------------------------- 42
3. Measurement of cellular viability --------------------------------------------------------- 42
4. Western blotting ----------------------------------------------------------------------------- 43
5. Establishment of the stable cell lines expressing GFP-LC3 and the fluorescence specifically in mitochondria or endoplasmic reticulum ------------------------------ 43
6. Establishment of the stable breast cancer cells overexpressing MnSOD or catalase --
------------------------------------------------------------------------------------------------ 44
7. Transfection ---------------------------------------------------------------------------------- 44
8. Small interfering RNAs -------------------------------------------------------------------- 45
9. Transmission electron microscopy -------------------------------------------------------- 46
10. Construction of the expression vector encoding AIP-1/Alix ------------------------- 46
11. Measurement of ROS and mitochondrial superoxide production ------------------- 46
12. Measurement of cytosolic and mitochondrial Ca2+ levels ---------------------------- 47
13. Flow cytometry for the analysis of DNA contents ------------------------------------ 47
14. Immunocytochemistry -------------------------------------------------------------------- 47
15. Measurement of intercellular level of reduced glutathione (GSH) ----------------- 48
16. 20S proteasome activity assay in intact cells ------------------------------------------ 48
17. Inhibition of purified 20S proteasome activity ---------------------------------------- 49
18. Human breast tumor xenograft experiments ------------------------------------------- 49
19. Clonogenic cell survival assay ----------------------------------------------------------- 50
20. Analysis of the effects of combinations of drugs -------------------------------------- 50
21. Statistical analysis ------------------------------------------------------------------------- 51
CHAPTER I. Superoxide anion and proteasomal dysfunction contributes to curcumin-induced paraptosis of malignant breast cancer cells ----------------------------------------------------------------------------------------------- 52
ABSTRACT ------------------------------------------------------------------------------------- 53
1. INTRODUCTION -------------------------------------------------------------------------- 55
2. RESULTS ------------------------------------------------------------------------------------- 57
2.1. The selective cytotoxic effects of curcumin on malignant breast cancer cells are not associated with apoptosis or autophagy --------------------------------------------- 57
2.1.1. Curcumin induces non-apoptotic cell death in malignant breast cancer cells--57
2.1.2. Curcumin induces non-autophagic cell death in malignant breast cancer cells ---------------------------------------------------------------------------------------------- 64
2.2. Curcumin induces paraptosis accompanied by swelling and fusion of mitochondria or ER in malignant breast cancer cells --------------------------------------------------- 69
2.3. Proteasomal dysfunction contributes to curcumin-induced paraptosis ------------ 87
2.4. Mitochondrial superoxide triggers curcumin-induced paraptosis ------------------ 94
3. DISCUSSION ------------------------------------------------------------------------------- 104
CHAPTER II. Simultaneous mitochondrial Ca2+ overload and proteasomal inhibition are responsible for the induction of paraptosis in malignant breast cancer cells --------------------------------------------- 119
ABSTRACT ------------------------------------------------------------------------------------ 120
1. INTRODUCTION ------------------------------------------------------------------------- 121
2. RESULTS ----------------------------------------------------------------------------------- 123
2.1. Curcumin induces paraptosis selectively in malignant breast cancer cells ------ 123
2.2. Mitochondrial Ca2+ overload is critically involved in curcumin-induced paraptosis --------------------------------------------------------------------------------------------- 129
2.3. Mitochondrial Ca2+ influxes act at the earliest stage of curcumin-induced paraptosis ---------------------------------------------------------------------------------- 143
2.4. Simultaneous mitochondrial Ca2+ overload and proteasomal dysfunction induce paraptosis in malignant breast cancer cells ------------------------------------------- 150
3. DISCUSSION ------------------------------------------------------------------------------- 168
CHAPTER III. Dimethoxycurcumin more effectively induces paraptosis than curcumin in breast cancer cells via stronger proteasomal inhibition and CHOP induction --------------------------------- 175
ABSTRACT ------------------------------------------------------------------------------------ 176
1. INTRODUCTION ------------------------------------------------------------------------- 178
2. RESULTS ----------------------------------------------------------------------------------- 179
2.1. DMC demonstrates more potent anti-cancer effect on breast cancer cells in vitro and in vivo --------------------------------------------------------------------------------- 180
2.2. DMC induces paraptosis in malignant breast cancer cells ------------------------- 185
2.3. DMC inhibits the proteasomal activity and induces CHOP expression more potently than curcumin -------------------------------------------------------------------- 197
2.4. DMC does not inhibit proteasomal activity and cellular viability in normal breast cells ---------------------------------------------------------------------------------------- 208
3. DISCUSSION -------------------------------------------------------------------------------213
III. CONCLUSION ---------------------------------------------------------------------- 226
REFERENCES ---------------------------------------------------------------------------- 230
국문요약 --------------------------------------------------------------------------------------- 261
