유방암 세포에서 ER-associated degradation 억제를 통한 Eeyarestatin1의 paraptosis 유도
Eeyarestatin1 induced paraptosis in breast cancer cells via inhibition of ER-associated degradation
- 주제(키워드) Eer1 , breast cancer , endoplasmic reticulum , mitochondria , paraptosis , ERAD , eIF2α
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024966
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Endoplasmic reticulum-associated protein degradation (ERAD) is a cellular pathway which targets misfolded protein of the endoplasmic reticulum (ER) for ubiquitination and subsequent degradation by proteasome. In this study, I investigated the anti-tumor effects of Eeyarestatin1 (Eer1), a potent inhibitor of ERAD, in various breast cancer cells. I found that treatment of breast cancer cells with Eer1 induced the dilation of mitochondria and the ER without any apoptotic characteristics. Prior to cell death, Eer1 treatment induced the accumulation of poly-ubiquitinated proteins as well as the proteins associated with ER stress and Eer1-induced accumulation of these proteins was completely blocked by cycloheximide. In addition, Eer1 treatment induced the activation of both JNKs and ERKs. Taken together, these results suggest that Eer1 treatment kills breast cancer cells via induction of paraptosis. Furthermore, I found that following Eer1 treatment the phosphorylation levels of eIF2α at Serine 51 were progressively reduced and pretreatment with salubrinal, an inhibitor of eIF2α phosphatase, markedly attenuated Eer1-induced vacuolation and cell death. In addition, overexpression of the non-phosphorylatable mutant eIF2α (with a mutation at serine 51 to alanine; S51A) markedly delayed them. Collectively, these results indicate that eIF2α dephosphorylation may critically contribute to Eer1-induced paraptosis in breast cancer cells.
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ABSTRACT -------------------------------------------------------------------------------------- i
TABLE OF CONTENTS ---------------------------------------------------------------- iii
LIST OF FIGURES ------------------------------------------------------------------------- v
I. INTRODUCTION ------------------------------------------------------------------------ 1
II. MATERIALS AND METHODS ------------------------------------------------- 5
A. Chemicals and antibodies --------------------------------------------------------------- 5
B. Cell culture --------------------------------------------------------------------------------- 5
C. Examination of changes the ER and mitochondria using stable cell lines expressing the fluorescence specifically in endoplasmic reticulum or mitochondria ------------------------------------------------------------------------------- 6
D. Cell viability assay (Live & Dead assay) --------------------------------------------- 6
E. Western blotting --------------------------------------------------------------------------- 7
F. Immunocytochemistry (ICC) ----------------------------------------------------------- 7
G. Transfection--------------------------------------------------------------------------------- 8
H. Small interfering (si) RNA -------------------------------------------------------------- 8
I. Overexpression of the eIF2α or eIF2α variant--------------------------------------- 9
J. Transmission electron microscopy (TEM) ------------------------------------------- 9
K. Statistical analysis ----------------------------------------------------------------------- 10
III. RESULTS --------------------------------------------------------------------------------- 11
1. Eeyarestatin-1 (Eer1) has an anti-cancer effect on various breast cancer cells via non-apoptotic and non-autophagic cell death --------------------------------- 11
2. Eer1 induces paraptosis accompanied by vacuolation of mitochondria and ER in malignant breast cancer cells ------------------------------------------------- 18
3. Accumulation of misfolded proteins within the ER via ERAD critically contribute to Eer1-induced vacuolation -------------------------------------------- 30
4. Eer1 induces ER stress and decrease in eIF2α phosphorylation critically contributes to Eer1-induced paraptosis--------------------------------------------- 32
5. Eer1 induces paraptosis selectively in breast cancer cells, sparing normal breast cells -------------------------------------------------------------------------------- 45
IV. DISCUSSION ---------------------------------------------------------------------------- 49
V. REFERENCES --------------------------------------------------------------------------- 61
