Disruption of thiol proteostasis sensitizes cancer cells to proteasome inhibitor-mediated paraptosis
- 주제(키워드) the thioredoxin system , glutathione , paraptosis , proteasome inhibitor
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032203
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Although the proteasome inhibitor (PI), bortezomib (Bz), is currently used as a front-line treatment for multiple myeloma, its clinical efficacy in solid tumors has not been satisfactory. In this study, I show that the cytotoxicity of various PIs, including Bz and carfilzomib, in breast cancer cell lines can be effectively enhanced not only by inhibition of the thioredoxin (Trx) system (including Trx 1 and Trx reductase 1) or glutathione reductase (GSR) through their silencing and pharmacological inhibition. I found that dual inhibition of proteasome and the Trx system or GSR induces paraptosis-associated cell death, which is accompanied by severe ER (endoplasmic reticulum) stress and ER-derived vacuolation. Interestingly, sensitization of Bz-mediated paraptosis-like cell death by inhibition of the Trx system or GSR was effectively blocked by various thiol-containing antioxidants, in an independent manner of ROS generation. In addition, a dual inhibition of proteasome and the Trx system or GSR reduced the ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG). Dithiothreitol (a reducing agent) or nicotinamide (a precursor of nicotinamide adenosine dinucleotide (NAD+) or nicotinamide adenosine dinucleotide phosphate (NADP+)) recovered cellular GSH/GSSG ratio and inhibited the paraptosis induced by Bz plus the Trx system or GSR inhibitors. GSH imbalance induced by inhibition of the Trx system or GSR under the proteasome-inhibiting condition may aggravate the accumulation of misfolded thiol-containing proteins and increase their S-glutathionylation, contributing to proteotoxicity-mediated paraptosis in cancer cells.
more초록/요약
현재 프로테아좀 억제제인 bortezomib (Bz)을 다발성 골수종 환자의 치료제로써 임상적으로 사용하고 있지만, 고형암 환자들에게는 항암 효과가 만족스럽지 못하다. 본 연구는 고형암 세포주에서 thioredoxin (Trx)과 thioredoxin reductase (TrxR)을 포함하는 Trx 시스템과 glutathione reductase (GSR)를 억제함으로써 Bz 또는 carfilzomib (Cfz)와 같은 proteasome 억제제의 항암효과를 paraptosis 유도를 통해 증진시킬 수 있음을 밝혔다. Paraptosis는 non-apoptotic 세포 사멸 모드 중 하나로 소포체 스트레스와 소포체 vacuolation을 동반하는 특징을 가진다. Trx 시스템과 프로테아좀 동시 억제 또는 GSR과 프로테아좀의 동시 억제로 인해 유도되는 paraptosis는 활성산소의 발생과는 무관하며, thiol을 포함하는 항산화제에 의해 효과적으로 억제된다. 게다가, Trx 시스템 억제제와 프로테아좀 억제제 및 GSR 억제제와 프로테아좀 억제제의 병합 처리는 환원된 glutathione (GSH)/산화된 glutathione (GSSG)의 비율을 감소시킨다. 환원제인 dithiothreitol (DTT)나 GSH환원에 필요한 nicotinamide (NAM)를 병합처리와 동시에 처리하면 GSH/GSSG 비율 감소를 복구시키고 paraptosis 유도를 효과적으로 억제한다. 따라서 Trx 시스템과 프로테아좀의 동시 억제 또는 GSR과 프로테아좀의 동시 억제는 GSH/GSSG의 불균형을 통해 산화적 스트레스를 유도하고, 결과적으로 단백질의 thiol잔기의S-glutathionylation을 유도하거나 thiol잔기를 갖는 단백질의 misfolding을 통해 소포체 스트레스와 소포체 팽창을 야기할 수 있다. 궁극적으로 세포 내의 단백질 항상성의 붕괴가 유도되어 단백질 독성 스트레스를 가중시켜paraptosis로 인한 세포 사멸이 나타날 수 있다.
more목차
I. Introduction 1
II. Materials and Methods 10
A. Chemicals and Antibodies 10
B. Cell Culture 11
C. Cell Viability Assay 11
D. Morphological Examination of The ER and Mitochondria 12
E. Immunoblot Analyses 12
F. Measurement of Proteasome Activity Employing UbG76V-GFP 13
G. Small interfering RNA- or small hairpin RNA-mediated knockdown of proteins 13
H. Measurement of ROS generation 14
I. Detection of intracellular GSH level 14
J. RNA isolation and quantitative real-time RT-PCR (qRT-PCR) 15
K. Isobologram Analysis 16
L. shRNA generation 16
M. Immunofluorescence Microscopy 17
N. Statistical Analysis 18
III. Results 19
Part1. Auranofin induces paraptosis via simultaneous inhibition of the thioredoxin reductase and proteasome in breast cancer cells. 19
1. Introduction 19
2. Results 25
2.1. Auranofin triggers paraptosis in breast cancer cells 25
2.2. AF-induced paraptosis requires the inhibition of both thioredoxin reductase and proteasome 31
2.3. Combining of AF and Bz at their sub-lethal doses spares non-transformed cells, in contrast to the effect of high-dose AF 38
2.4. AF plus PI of subtoxic doses synergistically induces paraptosis in breast cancer cells 40
2.5. GSH depletion rather than ROS generation is critical for the paraptosis induced by the dual inhibition of the thioredoxin reductase and proteasome 44
2.6. ATF4 upregulation critically contributes to the paraptosis induced by the dual inhibition of TrxR1 and proteasome 50
2.7. Upregulation of the ATF4/CHAC1 axis is critical for the paraptosis induced by the dual inhibition of the thioredoxin reductase and proteasome by degrading intracellular glutathione 54
Part2. Inhibition of the thioredoxin system overcomes cancer cells' resistance to Bz-mediated cytotoxicity by inducing paraptosis. 60
1. Introduction 60
2. Results 65
2.1. Bortezomib induces apoptosis in bortezomib-sensitive breast cancer cells 65
2.2. The expression of the thioredoxin system in breast cancer cells is differentially regulated depending on their Bz sensitivity 68
2.3. TrxR1 inhibitor plus the proteasome inhibitor also synergistically induce vacuolation-associated cell death in Bz-less sensitive breast cancer cells 75
2.4. Simultaneous inhibition of Trx system and proteasome induces vacuolation-associated cell death also in other Bz-less sensitive cancer cells but not in non-transformed cells 78
2.5. Simultaneous inhibition of the Trx system and proteasome induces paraptosis in cancer cells 83
2.6. Decrease in GSH/GSSG ratio plays a critical role in the paraptosis induced by inhibition of the thioredoxin system and proteasome 89
Part3. Inhibition of glutathione reductase sensitizes breast cancer cells to bortezomib-mediated cell death by disrupting protein thiol redox homeostasis. 100
1. Introduction 100
2. Results 102
2.1. Inhibition of the glutathione system effectively sensitizes breast cancer cells to PI-mediated cell death by inducing paraptosis 102
2.2. Thiol-containing antioxidants completely block the cell death induced by the glutathione reductase inhibition plus bortezomib. 113
2.3. Glutathione imbalance induced by glutathione reductase inhibition plus bortezomib may critically contribute to paraptosis. 116
2.4. The ATF4/CHAC1 axis is not involved in the paraptosis induced by glutathione reductase inhibition plus bortezomib. 123
IV. Discussion 126
V. Reference 136
국문 요약 163
