Lercanidipine 처리시 프로테아좀 억제제의 암세포 사멸을 증대시키는 paraptosis 세포 사멸 기전 연구
Lercanidipine enhances the cytotoxicity of bortezomib in cancer cells by inducing paraptosis-associated cell death
- 주제(키워드) Bortezomib , Lercanidipine , ER stress , Mitochondrial Ca2+ overload , Paraptosis
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029322
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Bortezomib (Btz), a proteasome inhibitor (PI), is effective in treating multiple myeloma and mantle cell lymphoma, but its anticancer effects are not satisfactory in solid tumors. In this study, we examined the possibility that the sensitivity to PIs can be enhanced by drug repositioning. We first show that lercanidipine (Ler), an antihypertensive agent, increases the cytotoxicity of various PIs including Btz, carfilzomib (Cfz), and ixazomib (Ixz) by inducing paraptosis-associated cell death, which is accompanied by severe dilation of mitochondria and the endoplasmic reticulum (ER) and in many solid tumor cell lines. We have found that mitochondrial Ca2+ overload induced by the combination of Btz and Ler is crucial for mitochondrial vauolation and subsequent mitochondrial membrane potential loss in MDA-MB 435S cells. In addition, Ler enhances Btz-mediated ER stress due to accumulation of misfolded proteins, contributing to ER vacuolization. Taken together, our results suggest that the combined regimen of PIs and Ler can effectively kill cancer cells through structural and functional disturbance in mitochondria and the ER.
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ABSTRACT i
TABLE OF CONTENTS ii
LIST OF FIGURES iv
LIST OF TABLES vii
I. INTRODUCTION 1
II. MATERIALS AND METHODS 17
A. Chemicals and antibodies 17
B. Cell culture 18
C. PI staining for cell viability assay 18
D. Determination of cell viability by MTT assay 19
E. Examination of morphologies of mitochondria and the ER employing the plasmids to
specifically label the ER or mitochondria 19
F. Western blotting 19
G. Immunocytochemistry 20
H. Measurement of cytosolic and mitochondrial Ca2+ levels 20
I. Isobologram analysis 21
J. Statistical analysis 21
III. RESULTS 23
1. Various dihydropyridine effectively enhance Btz-mediated cell death in breast cancer cells 23
2. Lercanidipine effectively enhances PI-mediated cell death in a various cancer cells 27
3. Combined treatment with Ler and Btz induces non-apoptotic, non-necroptotic, and non-
autophagic cell death 33
4. Combination of Ler and Btz induces paraptosis 40
5. Combined treatment with Ler and Btz enhances ubiquitinated protein accumulation and ER
stress 47
6. Combination of Ler and Btz disrupts Ca2+ homeostasis via mitochondrial Ca2+ overload 50
7. Mitochondrial Ca2+ uptake critically contributes to Ler/Btz-induced paraptotic cell death 56
IV. DISCUSSION 59
V. REFERENCES 66
국문요약 - 79
