Cyclosporine A sensitizes breast cancer cells to bortezomib by inducing paraptosis
- 주제(키워드) Cyclosporine A , ER stress , Ca2+ , ROS , Paraptosis
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030233
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Cyclosporine A (CsA), a natural product extracted from the Tolypocladium inflatum, is known to have immunosuppressive and anti-fungal activities. In recent studies, CsA was reported to have an anti-cancer activity in breast, colon, and cervical cancer cells, but its precise underlying mechanism is not fully understood. In the present study, I found that treatment of MDA-MB 435S cells with CsA induces cytoplasmic vacuolation originated mainly from mitochondria and partially from the endoplasmic reticulum (ER), leading to growth arrest, rather than cell death. In contrast, the combination of bortezomib and CsA (Bz/CsA) accelerates cell death through the massive cytoplasmic vacuolation derived initially from mitochondria and subsequently from the ER in these cells. Bz/CsA-induced cell death as well as dilation of both mitochondria and the ER was significantly inhibited by cycloheximide, but not by the inhibitors of apoptosis, necroptosis, or autophagy, suggesting that Bz/CsA induced paraptotic cell death. I found that CsA dramatically enhances Bz-mediated ER stress. In addition, Bz/CsA triggered mitochondrial Ca2+ overload, and RU360, a specific inhibitor of mitochondrial calcium uniporter (MCU), effectively inhibited the cytoplasmic vacuolation and subsequent cell death by Bz/CsA. Furthermore, I found that reactive oxygen species (ROS) were generated in the process of mitochondria- and ER-derived vacuolation and pretreatment with antioxidants significantly attenuated Bz/CsA-induced cell death. Collectively, these results suggest that mitochondrial Ca2+ overload, enhanced ER stress, and ROS are responsible for the induction of Bz/CsA-induced paraptosis in breast cancer cells.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
A. Chemicals and antibodies 6
B. Cell culture 7
C. Measurement of cellular viability (Live & Dead assay) 7
D. Clonogenic assay 7
E. Immunoblotting 8
F. Morphological examination of the ER and mitochondria 8
G. Immunofluorescence microscopy 9
H. Isobologram analysis 9
I. Transmission electron microscopy 10
J. Measurement of intracellular Ca2+ levels 10
K. Measurement of intracellular reactive oxygen species (ROS) levels 11
L. Small interfering RNA mediated knockdown of the genes 11
M. Statistical analysis 11
III. RESULTS 13
1. CsA was identified as a dilating agent of mitochondria and a Bz sensitizer 13
2. Treatment with CsA alone inhibits cellular growth, but it dose not effectively kill MDA-MB 435S cells 17
3. Combination of Bz and CsA effectively induces paraptotic cell death in breast cancer cells 23
4. Ca2+ imbalance due to mitochondrial Ca2+ overload critically contributes to Bz/CsA-induced paraptosis 37
5. ROS generation partially contributes to the cytotoxic effect of Bz/CsA without affecting dilation of mitochondria and the ER 46
6. Perturbation of mitochondrial dynamics enhances Bz/CsA-induced paraptosis 52
IV. DISCUSSION 63
V. REFERENCES 74
