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프로테아좀 및 리소좀 억제를 통해 뇌종양 세포 사멸을 증진시키는 방안에 관한 기전 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 최경숙 | - |
| dc.contributor.author | Kim inyoung | - |
| dc.date.accessioned | 2019-10-21T07:27:51Z | - |
| dc.date.available | 2019-10-21T07:27:51Z | - |
| dc.date.issued | 2016-08 | - |
| dc.identifier.other | 23335 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/18862 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2016. 8 | - |
| dc.description.tableofcontents | I. INTRODUCTION 1 II. MATERIALS AND METHODS 24 A. MATERIALS 24 1. Chemicals 24 2. Antibodies 24 B. METHODS 25 a. Culture of glioma cell lines and normal human astrocytes 25 b. Determination of cellular viability using calcein-AM and EthD-1 (Live-Dead assay) 25 c. Determination of cellular viability by an MTT assay 26 d. Determination of synergism by the combination inducx (CI) method 26 e. Colonogenic cell survival assay 26 f. Western blotting 27 g. Establishment of the stable cell lines expressing the fluorescence specifically in endoplasmic reticulum 27 h. Immunocytochemisty 28 i. small interfering RNAs 28 j. Knockdown experiments using small hairpin (sh) RNA 29 k. Assay of aggresome formation 29 l. Visualization of autophagic vacuoles 29 m. LysoTracker Red staining 30 n. Detection of acidic vesicular organelles (AVOs) with acridine oranges staining 30 o. BODIPY FL-pepstatin staining 30 p. HCS LipidTOXTM neutral lipid staining 30 q. Live-cell FluoZin-3 experiments 31 r. Intracellular pH detection 31 s. Transmission electron microscopy 31 t. Measurement of mitochondrial superoxide production 32 u. Statistical analysis 32 PART1.eIF2a modulates bortezomib-induced vacuolation and cell death in glioma cells via phosphorylation-dependent and independent manner 33 1. INTRODUTION 34 2. RESULTS 40 2.1. Proteasome inhibitors indue non-apoptotic cell death in glioma cell death in glioma cells accompanying ER-derived vacuolation 40 2.2. The eIF2a phosphorylation at serin 51 blocks bortezomib-induced vacuolation and cell death in glioma cells 47 2.3. Protein synthesis may play an important role in ER vacuolation and cell death by bortezomib 52 2.4. Dephosphorylation of eIF2a rapidly accumulates aggregates in cells, leading to cell death by bortezomib 56 2.5. The eIF2a downregulation sensitizes glioma cells to bortezomib-induced apoptosis 58 2.6. Dephosphorylation and downregulation of eIF2a may be responsible for the synergistic effect of salubrinal and bortezomib on ER-derived vacuolation and cell death 61 2.7. Protein synthesis may play an important role in ER vacuolation and cell death by salubrinal and bortezomib 69 2.8. Bortezomib-induced poly-ubiquitination of proteins in further enhanced but ubiquitinated protein aggregates are dispersed by co-treatment with salubrinal 71 2.9. Human astrocytes are resistant to the combined treatment with salubrinal and bortezomib 75 3. DISCUSSION 77 PART2. Flunarizine overcomes the resistance of glioma cells to bortezomib via induction of paraptosis-like cell death 84 1. INTRODUTION 85 2. RESULTS 87 2.1. Flunarizine sensitizes glioma cells, but not normal astrocytes, to bortezomib-mediated non-apoptotic cell death 87 2.2. The combined treatment with flunarizine and bortezomib induces the swelling of ER in glioma cells 93 2.3. CHOP and NOXA is critically involved in the cell death by flunarizine plus bortezomib 99 2.4. Flunarizine plus bortezomib induces paraptosis-like cell death 101 2.5. Critical role of ROS in the cell death by the combined treatment with flunarizine and bortezomib 103 2.6. Critical role of intracellular Ca2+ in the cell death by the combined treatment with flunarizine and bortezomib 105 2.7. Disruption of aggresome formation may be involved in the cell death induced by flunarizine and bortezomib 107 3. DISCUSSION 110 PART3. Combination of chloroquine and kaempferol induces cell death via lysosomal overloading and rupture 116 1. INTRODUTION 117 2. RESULTS 120 2.1. Kaempferol sensitizes human glioma cells to chloroquine-mediated cell death, but not normal astrocytes 120 2.2. Kaempferol promotes autophagy in glioma cells 124 2.3. The sensitizing effect of chloroquine on kaempferol-mediated cell death may be associated with its activity as the lysosomal inhibitor 129 2.4. Co-treatment with kaempferol disrupts chloroquine-induced swollen lysosomal structures 134 2.5. Cathepsin D released from the disrupted ysosomes may contribute to the cell death by the combined treatment with kaempferol and chloroquine 140 2.6. Lysosomal rupture by combined treatment with kaempferol and chloroquine injures cellular organelle including mitochondrial and DNA 147 3. DISCUSSION 152 IV. CONCLUSION 160 V. REFERENCES 164 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 프로테아좀 및 리소좀 억제를 통해 뇌종양 세포 사멸을 증진시키는 방안에 관한 기전 연구 | - |
| dc.title.alternative | Mechanistic study on the therapeutic strategy to enhance glioma cell death through inhibition of proteasome and lysosome | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | In Young Kim | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2016. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 758568 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023335 | - |
| dc.subject.keyword | proteostasis | - |
| dc.subject.keyword | protein degradation | - |
| dc.subject.keyword | bortezomib (BZ) | - |
| dc.subject.keyword | chloroquine (CQ) | - |
| dc.subject.keyword | eIF2a | - |
| dc.subject.keyword | endoplasmic reticulum (ER) vacuolation | - |
| dc.subject.keyword | lysosomal rupture | - |
| dc.description.alternativeAbstract | Malignant gliomas, the most common brain tumors with high mortality, remain largely incurable despite multimodal treatments including surgical resection, radiotherapy, and chemotherapy. Thus, the researchers are currently attempting to develop novel therapeutic strategies for malignant gliomas. Recent studies show that dysregulation of protein maintenance has been considerably linked to many diseases. Therefore, protein degradation system has become a platform for drug targeting, and mechanism-based drugs are currently developed. Bortezomib (BZ), a proteasome inhibitor, and chloroquine (CQ), a lysosomotropic agent, can disrupt cellular protein homeostasis (proteostasis) via interruption of protein degradation systems, including proteasome and lysosome. In part I, we investigate the role of eIF2a in BZ-induced cell death. We found that ER-derived dilation preceded bortezomib induced cell death. eIF2α was initially phosphorylated but it was dephosphorylated in parallel with the ER dilation. Overexpression of eIF2a WT markedly increased rate of phosphorylated eIF2a (exogenous) and blocked bortezomib-induced ER vacoulation and cell death. In contrast, overexpression of dominant negative eIF2a S51A does not affect rate of phosphorylated eIF2a and cell death. These results suggests that dephosphorylation of eIF2a may be important for the ER dilation and cytotoxic effect of bortezomib. These results suggest that eIF2a dephosphorylation rather than its phosphorylation may be important for the cytotoxic effect of bortezomib. In addition, knockdown of eIF2a sensitized bortezomib-induced cell death via increasing caspase-dependent apoptosis. Collectively, regulation of eIF2a phosphorylation and expression may provide a therapeutic strategy to sensitize glioma cell to bortezomib-mediated cell death. We found that combined treatment with bortezomib and salubrinal, an inhibitor of GADD34-PPiC phosphatase complex, very effectively induced the dilation of the endoplasmic reticulum and subsequent cell death. Interestingly, either salubrinal or bortezomib increased the phosphorylation levels of eIF2α, but combined treatment markedly reduced them. In addition, co-treatment with salubrinal decreased expression levels of eIF2a. Taken together, these results suggest that salubrinal effectively overcomes the resistance of malignant glioma cells to BZ, via dependently- and independent-manner of eIF2a phosphorylation. In part II, we investigate whether co-treated phytochemicals sensitized CQ-induced cell death in glioma cells. We show that treatment with subtoxic doses of CQ, when combined with kaempferol, a flavonoid, effectively induces cell death in various glioma cells, but not in normal astrocytes, suggesting that the combined regimen using kaempferol and CQ may provide a safe therapeutic strategy to selectively kill resistant glioma cells. The cell death induced by kaempferol and CQ in U251MG cells was partially dependent on caspase-mediated apoptosis and accompanied by mitochondrial dysfunction, ER stress, and DNA damage. We found that kaempferol treatment increased the numbers of lysosome, whereas CQ treatment increased the lysosomal masses leading to their swelling. Combined treatment with kaempferol and CQ further increased the lysosomal masses, but culminated in the disruption of lysosomal compartments. While kaemfperol treatment increased the active mature forms of cathepsin D, CQ treatment markedly blocked the processing and activity of cathepsin D. Interestingly, combined treatment induced the release of unprocessed cathepsin D into the cytosol and the recovery of its activity. Knockdown of cathepsin D significantly attenuated the cell death induced by kaempferol and CQ, suggesting the functional involvement of these released cathepsin D proteins in this cell death. Taken together, our results suggest that the membrane destabilization of accumulated lysosomes and the resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by kaempferol plus CQ. In this study, we show that salubrinal effectively overcomes the resistance of malignant glioma cells to BZ, via dependently- and independent-manner of eIF2a phosphorylation. Also, we show that combined treatment with kaempferol and CQ effectively induces cell death in glioma cells, via lysosomal overloading and rupture. Collectively, these results suggest that proteostasis perturbation by targeting of UPR components or lysosome may provide potential therapeutic effect for targeting malignant glioma cells over normal cells. | - |
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