Curcumin에 의한 악성 유방암 세포 선택적인 non-apoptotic 세포 사멸 유도 기전
Curcumin induces non-apoptotic cell death selectively in malignant breast cancer cells
- 주제(키워드) Breast cancer , Curcumin , Paraptosis , ROS , Ca^(2+) , 유방암 , 활성산소종
- 발행기관 아주대학교 일반대학원
- 지도교수 최경숙
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Malignant breast cancers are resistant to various pro-apoptotic therapies, such as radiotherapy and conventional chemotherapy. In this study, we examined the effect of curcumin, a polyphenolic compound from the rhizome of Curcuma longa, in various human breast cancer cells and mammary epithelial cells. Treatment with curcumin preferentially cytotoxic to highly invasive breast cancer cells such as MDA-MB 231, MDA-MB 435S and Hs578T cells over weakly invasive T-47D, MCF-7 and BT-483 cells. In contrast, normal breast MCF-10A and human mammary epithelial cells were comparatively resistant to curcumin treatment. Therefore, treatment with curcumin may be a safe and effective strategy for treating malignant breast cancer cells. During curcumin-induced cell death in malignant breast cancer cells, apoptotic features, including cell shrinkage and caspase activation, were not observed. Although significant vacuolation preceded curcumin-induced cell death, autophagic features, including autophagosomal or autophagolysosomal structures and translocation of LC3 into vacuoles, were not observed. We found that curcumin-induced vacuolation was derived from the swelling of mitochondria and endoplasmic reticulum. Furthermore, curcumin-induced vacuolation and subsequent cell death in malignant breast cancer cells was significantly attenuated by a transcription inhibitor actinomycin D, a translation inhibitor cycloheximide, suppression of ERK2 expression by its small interfering RNA or JNK inhibitor. At the early phase of curcumin treatment, high levels of superoxide anion were generated and overexpression of MnSOD, but not Cu/ZnSOD or catalase, significantly blocked curcumin-induced paraptosis. We also found that Ca^(2+) was massively accumulated in swollen mitochondria of curcumin-treated malignant breast cancer cells. Pretreatment with ruthenium red, an inhibitor of mitochondrial Ca^(2+) uniporter, or dantrolene, ryanodine receptor (RyR) channel inhibitor, completely blocked both curcumin-induced vacuolation and subsequent cell death, suggesting that abrupt increase in mitochondrial Ca^(2+) levels may play a critical role in curcumin-induced paraptosis in malignant cancer cells. Taken together, superoxide anion generated by curcumin may trigger the mitochondrial damage and abrupt accumulation of Ca^(2+) in mitochondria, leading to paraptosis in malignant breast cancer cells.
more초록/요약
Apoptosis에 대한 내성을 획득한 악성 유방암은 방사선 치료법이나 화학 항암 요법의 치료 효과가 좋지 않으므로 non-apoptotic한 세포 사멸 모드의 유도를 통해 항암 효과를 도모하는 것이 바람직할 수 있다. Curcumin은 다년생 식물인 심황의 뿌리에 다량 함유되어 있는 curcuminoid라고 불리는 polyphenol 색소 화합물의 주성분으로 항산화, 항염증, 암 예방 및 암 치료 능력을 갖는다고 알려져 있다. 본 연구에서는 curcumin 처리 시 정상 세포는 별 영향을 받지 않는 반면, 악성 유방암 세포에서는 현저하게 세포 사멸이 유도되므로 curcumin 처리는 안전하면서도 효과적인 악성 유방암 치료책으로써의 가능성을 보였다. Curcumin을 처리한 악성 유방암 세포에서는 apoptosis나 autophagy의 특징들이 관찰되지 않았으며, 대신 세포 사멸 전에 mitochondria와 ER swelling이 선행되었다. 또한 세포 사멸 시 transcription과 translation 과정을 필요로 하며, ERK2 와 JNK의 활성을 억제한 경우 curcumin에 의한 세포 사멸이 억제되었으므로 curcumin은 악성 유방암 세포에서 “paraptosis”라 불리는 새로운 세포 사멸 모드를 유도한다고 볼 수 있다. 신호전달 과정으로는 초기에 발생되는 활성산소종과 함께 Ca^(2+)이 ER의 ryanodine receptor Ca^(2+) channel을 통해 mitochondria 내로 축적되는 것이 curcumin에 의한 paraptosis에 중요하다는 것을 확인하였다. 본 연구에서 같이 curcumin에 의한 paraptosis 신호전달 연구는 향후 악성 유방암 치료 효과를 증진시키기 위한 표적 발굴에 중요한 단초를 제공할 것이다.
more목차
ASTRACT = i
TABLE OF CONTENTS = iii
LIST OF FIGURES = vi
I. INTRODUCTION = 1
II. MATERIALS AND METHODS = 6
A. Chemicals and antibodies = 6
B. Cell culture = 7
C. Measurement of cell viability = 8
D. Immunoblotting = 8
E. RT-PCR = 8
F. Establishment of the stable cell lines overexpressing Akt or pcDNA3 and the stable cell lines expressing the fluorescence specifically in mitochondria or endoplasmic reticulum (ER) = 10
G. Construction of viral vectors overexpressing Cu/ZnSOD, MnSOD or catalase and transduction = 10
H. Cellular morphology and chromatin condensation observation by 40,6-diamidino-2- phenylIndole (DAPI) staining = 11
I. Flow cytometric analysis = 11
J. GFP-LC3 translocation = 12
K. Small interfering (si) RNAs = 12
L. Measurement of mitochondrial membrane potential (MMP) = 13
M. Transmission electron microscopy = 13
N. Measurement of ROS = 14
O. Measurement of mitochondrial superoxide anion = 14
P. Measurement of ER stress = 14
Q. Measurement of intercellular level of reduced glutathione (GSH) = 14
III. RESULTS = 16
1. Curcumin induces non-apoptotic cell death selectively in highly invasive breast cancer cells, but not in weakly invasive and normal cells = 16
2. Curcumin induces ER stress-mediated non-apoptpotic cell death = 25
3. Curcumin induces non-autophagic cell death in highly invasive breast cancer cells = 32
4. Curcumin induces paraptosis accompanying the swelling of mitochondria and ER in highly invasive breast cancer cells = 36
5. Curcumin-induced mitochondrial superoxide production triggers mitochondrial damage and subsequent parpatosis = 46
6. Excessive increase in Ca2+ uptake in mitochondria is critical for curcumin-induced paraptosis = 54
7. Other effects of curcumin on various signaling molecules in breast cells = 60
(1) Various anti-apoptotic proteins = 60
(2) NF-kB and STAT3 = 66
(3) Intracellular GSH levels = 68
(4) CHOP = 75
IV. DISSCUSSION = 79
REFERENCES = 90
국문요약 = 114
