臺灣博碩士論文加值系統

槲黃素是一種常見於蔬果中的植物性生物黃酮，已經被用於預防慢性攝護腺炎和攝護腺癌病患，它被報導具有抗發炎、抗氧化和抗癌的作用，但其詳細的抗癌作用機轉並不被完全了解。藉由化學蛋白體學，結合了藥物親和層析和質譜儀的生物資訊分析來研究槲黃素的直接結合蛋白質，我們鑑定到hnRNPA1和槲黃素有交互作用，並由西方點墨法和表面電漿共振生物感測儀確定hnRNPA1與槲黃素交互作用的專一性。並更進一步，利用基因工程將hnRNPA1片段化並分別與槲黃素進行結合反應，我們發現槲黃素和hnRNPA1結合在C-terminal區域，阻礙了hnRNPA1往返細胞核質的正常功能，造成hnRNPA1 堆積在細胞質中，並暫存在stress granules，無法進行正常功能下抗凋亡蛋白質cIAP1 mRNA的轉譯，使得細胞最終走向凋亡。這是第一篇報導hnRNPA1是槲黃素的直接作用蛋白質，並闡釋了槲黃素的抗攝護腺癌機轉之一是透過結合hnRNPA1，影響了細胞內抗凋亡蛋白質的平衡造成癌細胞的死亡。

Quercetin, a flavonoid abundantly present in plants, is widely used as a phytonutrient in prostatitis and prostate cancer. Although quercetin has been reported to have a number of beneficial effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and surface plasmon resonance experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules (SGs) after treatment of cells with quercetin for up to 48 hours, and the levels of cellular inhibitor of apoptosis protein-1 (cIAP1), an IRES-translation dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1—insights that were obtained using a chemical proteomics strategy.

口試委員會審定書 2
中文摘要 2
Abstract 4
Abbreviations 5
Table of Contents 7
Chapter I – Overview and Rationale 10
1.1 Prostate cancer 11
1.2 Therapeutics in Prostate Cancer 14
1.3 Introduction of quercetin and its applications on prostate cancer 15
1.4 Molecular targets of quercetin 17
1.5 Introduction of chemical proteomics 18
1.6 Specific aim 19
Chapter II –Identification of hnRNPA1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells 20
2.1 Workflow for identification and characterization of quercetin-binding proteins 21
2.2 Experimental Procedures 21
Materials 21
Clones and constructs 22
Cell culture and transient transfection 23
Cell treatment and sample preparation 24
Quercetin uptake assay 24
Cell viability assay 25
Cell apoptosis assays 25
Affinity chromatography 26
In-gel digestion, MS analysis and protein identification 26
Immunoblotting 27
KD measurement by SPR 28
Immunofluorescence analysis 29
Immunoprecipitation 30
Subcellular Fractionation 30
Ribonucleoprotein Immunoprecipitation (RIP) and quantitative RT-PCR 31
Statistical analysis 31
2.3 Results 33
Time course of quercetin uptake, cell viability, and apoptosis in quercetin-treated PC-3 cells 33
Analysis of quercetin-binding proteins 33
Kinetic analysis of quercetin-hnRNPA1 interaction 34
Cytoplasmic accumulation of hnRNPA1 and reduced Tnpo1 binding after quercetin treatment 35
The mechanism underlying the quercetin-induced change in hnRNPA1 localization in PC-3 cells is independent of F-peptide phosphorylation 36
Cytoplasmic accumulation of hnRNPA1 after quercetin treatment results in greater IRES-dependent mRNA binding but diminished protein expression 38
2.4 Discussion 40
Potential quercetin binding proteins 40
Regulatory role of hnRNPA1 in quercetin-induced apoptosis 42
Possible binding site of quercetin at C-terminal end of hnRNPA1 45
Chemical proteomics approaches in drug discovery 45
Chapter III –Conclusion and perspectives 49
Conclusion and perspectives 50
List of Tables, Figures and Instrument 52
Table I. Identification of quercetin-binding proteins by LC MS/MS. 53
Table II. Primers used in cloning and qPCR studies. 54
List of instrument 55
Figure 1. Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. 56
Figure 2. Quercetin uptake in quercetin-treated PC-3 cells. 57
Figure 3. Cell viability in quercetin-treated PC-3 cells. 58
Figure 4. Cell apoptosis in quercetin-treated PC-3 cells. 59
Figure 5. Identification and validation of quercetin binding targets using affinity chromatography and immunoblotting. 60
Figure 6. Validation of quercetin binding targets using SPR binding assays. 61
Figure 7. Cytoplasmic accumulation of hnRNPA1 after quercetin treatment. 62
Figure 8. Reduced Tnpo1 binding after quercetin treatment. 63
Figure 9. Modification verification of F-peptides of cytoplasmic hnRNPA1 by LC-MS/MS. 64
Figure 10. Mutant F-peptide has specific interaction with quercetin. 65
Figure 11. Localization of WT hnRNPA1 and F2mt-hnRNPA1 in transiently transfected PC-3 cells following quercetin treatment. 66
Figure 12. Cytoplasmic fraction of cIAP1 mRNA bound by hnRNPA1 67
Figure 13. Subcellular fractionation of PC-3 cells after quercetin treatment 68
Figure 14. Immunoblotting of quercetin-treated cells after knockdown of cIAP1 69
Figure 15. Apoptosis and viability analysis of quercetin-treated PC-3 cells with transient knockdown of hnRNPA1 and cIAP1. 70
Figure 16. Quercetin induces hnRNPA1 localization to SGs. 71
Figure 17. Model of quercetin binds to hnRNPA1 and induces apoptotic mechanism in PC-3 cells 72
References 73

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