三唑化合物作為自噬促進劑用於疾病治療

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(1)國立臺灣師範大學生命科學系博士班研究生論文. 三唑化合物作為自噬促進劑用於疾病治療 Triazole derivatives as autophagy enhancers. 研究生：謝長亨 Chang Heng Hsieh. 指導教授：方剛博士 Kang Fang. 中華民國一○六年二月.

(2) 三唑化合物作為自噬促進劑用於疾病治療 Triazole derivatives as autophagy enhancers. 第一部分:三唑化合物用於治療麩醯胺酸擴增疾病的應用及機制. 第二部分:三唑化合物用於治療非小細胞肺癌細胞的應用及機制. Part-I. The application and mechanisms to treat polyglutamine diseases. Part-II. The application and mechanisms to treat human non-small cell lung cancer cells. 2.

(3) 誌謝. 本論文承【罕見疾病基金會財團法人】102 年第十五屆博碩士論文獎助學金支持，謹致謝忱. 3.

(4) 誌謝研究是一個縝密及複雜的工作，充滿技術與邏輯思考，這些都是我在進入研究所時嚴重缺乏的特質。進入師大這個大家庭後，使我在這培育師資的環境中受到薰陶下漸漸走上軌道。在求學的過程中有太多人要感謝，指導老師方剛教授充分的授權，讓我能將一個全新的主題，自由的從零開始架構出一個完整的結果，同時也適時地為我踩下煞車，並且在論述表達上充分的訓練我。許多授課老師諄諄教誨亦使我受用良多，尤其王慈蔚老師與林炎壽老師的教學下，大量的論文導讀及邏輯思考訓練令我在解決問題上有很大的助益。口試委員陳瓊美醫師與黃偉邦教授的建議讓我的論文更加完整，化學系姚清發教授提供實驗藥材與化學上相關的協助。同時得到學校其他老師們及系辦公室的助教們時時給予關心及幫忙，讓整個研究時光不會孤單也使阻礙減少。實驗室中感謝學長景平做為一個前導；俊彥學長的人生相談；中岳學長的支持與承勳學長的異國經驗；學姊亭潔細心的指導；蕙芳學姊作為嚴謹的榜樣；同學楷涵、雅筑共同營造良好研究氣氛；學妹采蓁、憶如和學弟偉賢一同為這新的主題付出努力；學妹玉玲、佩岑和學弟柏緯、潮永及文興也提供許多寶貴的意見。最後謝謝一路體諒包容我的家人及親友陪伴我能完成這個學位。. 4.

(5) INDEX INDEX···························································································································5 CHAPTER I ABSTRACT ·························································································11 CHAPTER II 中文摘要 ····························································································13 LIST OF ABBREVIATIONS ····················································································15 CHAPTER III AUTOPHAGY··················································································17 CHAPTER IV·············································································································19 PART I THE APPLICATION AND MECHANISMS TO TREAT POLYGLUTAMINE DISEASES··············································································19 I.. INTRODUCTION······························································································19. 1.. Protein aggregation diseases ··································································································19. 2.. Triazole derivatives and neurodegeneration ·········································································19. 3.. Autophagy and protein aggregation diseases ········································································20. II.. MATERIAL AND METHOD ·······································································21. 1.. Cell culture ····························································································································21. 2.. Plasmid and transfection ·······································································································21. 3.. Reagents·································································································································22. 4.. FACScalibur analysis ············································································································24. 5.. Immunoblotting ·····················································································································24. 6.. Quantification of aggregation dots ························································································25. 7.. Confocal microscopy detection ······························································································25. 5.

(6) 8.. III.. Statistical analysis··················································································································26. RESULTS········································································································27. 1.. Establishment of inducible polyQ stable cell lines ································································27. 2.. Autophagic enhancer screening·····························································································27. 3.. More evidence of OC-13-activated autophagy. ·····································································28. 4.. Amelioration of Q79-EGFP aggregates by OC-13 ································································29. 5.. The autophagic clearance is related to JNK signaling pathway activation ··························30. 6.. Clearance of the Q79-EGFP aggregates by JNK-mediated autophagy ································30. 7.. Exclusion of nucleus polyQ aggregation ···············································································31. 8.. Transient Httex1 polyQ transfection ·····················································································31. 9.. OC-13-induced autophagy in cells transfected with Httex1-Q97 ··········································32. 10.. Phosphorylation of JNK and Akt are increased by OC-13 in Httex1-Q97 transfected cells 33. 11.. OC-13 decreased Httex1-Q97 aggregations ··········································································33. 12.. Elimination of Httex1-Q97 aggregation by OC-13-mediated autophagy ·························34. 13.. Elimination of aggregates by JNK-mediated autophagy ··················································34. IV.. DISCUSSION ·································································································36. CHAPTER V ··············································································································40 PART II THE APPLICATION AND MECHANISMS TO TREAT HUMAN NON-SMALL CELL LUNG CANCER CELLS ·····················································40 I.. INTRODUCTION······························································································40. 1.. Lung cancer ···························································································································40. 2.. Triazole derivatives and cancer ·····························································································40. 6.

(7) 3.. Autophagic cell death ············································································································41. 4.. Cancer and autophagy ···········································································································41. II.. MATERIAL AND METHOD ·······································································42. 1.. Cell culture ····························································································································42. 2.. Plasmid and transfection ·······································································································43. 3.. Reagents·································································································································43. 4.. FACScalibur analysis ············································································································47. 5.. Immunoblotting ·····················································································································47. 6.. Animals and treatments ········································································································48. 7.. Histochemical staining ···········································································································48. 8.. Cell viability assay ·················································································································49. 9.. Colony forming assay ············································································································49. 10.. Statistical analysis ·············································································································49. III.. RESULTS········································································································51. 1.. BTO inhibited cell growth by apoptosis ················································································51. 2.. Enhancement of apoptosis and autophagy characters by BTO ············································51. 3.. Autophagy-mediated apoptosis by BTO ···············································································52. 4.. BTO increase ROS in A549 cells ···························································································52. 5.. BTO repressed growth in nude mice bearing xenograft tumors ···········································53. IV.. DISCUSSION ·································································································55. CHAPTER VI CONCLUSION ················································································58 CHAPTER VII FIGURES ························································································60. 7.

(8) Figure 1. Establishment of polyQ disease cell models. ···································································61 Figure 2. Screening of autophagy enhancing chemicals ·································································63 Figure 3. Changes of cell viability and aggregation intensities by candidate compounds. ············65 Figure 4. The variations of autophagy markers. ············································································67 Figure 5. The candidate chemical OC-13 and cell viability determination ····································68 Figure 6. Activation of lysosome by OC-13. ···················································································71 Figure 7. Induction of autophagy markers by OC-13. ···································································73 Figure 8. Inhibition of autophagy by Baf A1. ·················································································74 Figure 9. Autophagosome formation and aggregates clearance by OC-13. ···································76 Figure 10. Elimination of Q79 aggregation dots by OC-13. ···························································77 Figure 11. Elimination of the insoluble EGFP by OC-13. ······························································79 Figure 12. Activation of JNK pathway. ··························································································80 Figure 13. Elimination of Q79 aggregation by JNK pathway and autophagy. ······························82 Figure 14. Elimination of the insoluble EGFP as affected by inhibitors. ·······································83 Figure 15. The increase of autophagosomes by JNK pathway. ······················································85 Figure 16. Elimination of nucleus Q79 aggregation. ······································································88 Figure 17. Clearance of nucleus Q79 aggregation by autophagy and JNK pathway. ····················90 Figure 18. Establishment of Httex1 Q25 and Q97 cell models. ······················································91 Figure 19. Increasing of autolysosome by OC-13 in Httex1-Q25 and Q97-transfected cells. ·········93 Figure 20. Activation of lysosomes by OC-13 in Httex1-Q97 transfected cells. ······························95 Figure 21. Formation of autophagosome in Httex1-Q97 transfected cells. ····································96 Figure 22. Induction of autophagic markers in Httex1-Q97 transfected cells by OC-13. ··············98 Figure 23. Maintenance of cell viability in Httex1-Q97 transfected cells. ····································100. 8.

(9) Figure 24. Activation of JNK, Akt and S6K in Httex1-Q97 transfected cells. ······························101 Figure 25. Elimination of aggregations in Httex1-Q97 transfected cells. ·····································103 Figure 26. Elimination of the insoluble Httex1-Q97 aggregates by OC-13. ·································105 Figure 27. Clearance inhibition of aggregation. ···········································································107 Figure 28. Clearance inhibition of insoluble Httex1-Q97. ····························································109 Figure 29. Elimination of Httex1 aggregation dots by JNK pathway and autophagy. ················111 Figure 30. The increase of autophagosomes by JNK pathway in Httex1-Q97 transfected cells. ·112 Figure 31. Summary of OC-13 induced polyQ clearance by autophagy. ·····································113. FIGURE II ················································································································114 Figure 32. Inhibition of cell proliferation in human non-small cell lung cancer cells. ·················115 Figure 33. Activation of apoptosis by BTO.··················································································118 Figure 34. Activation of autophagy and apoptosis by BTO. ························································119 Figure 35. Apoptosis and autophagy in adenocarcinoma. ····························································121 Figure 36. Formation of autophagosome and autolysosome by BTO. ·········································123 Figure 37. Repression of autophagic flux by 3-MA. ·····································································125 Figure 38. Inhibition of BTO-mediated apoptosis by 3-MA. ·······················································127 Figure 39. BTO-induced autophagic apoptosis in A549. ······························································128 Figure 40. Increase of ROS intensity by BTO. ·············································································130 Figure 41. Inhibition of autophagy by NAC. ················································································132 Figure 42. Inhibition of tumor growth in nude mice xenografts. ·················································134 Figure 43. Increase of apoptosis in nude mice xenografts. ···························································136 Figure 44. Decrease of proliferation and increase of autophagy in tumor. ··································138 Figure 45. Promotion of apoptosis marker in tumors. ·································································140. 9.

(10) Figure 46. Summary of BTO induced autophagy. ········································································141. CHAPTER VIII REFERENCE ··············································································142 CHAPTER IX. APPENDIX ··················································································152. 10.

(11) Chapter I Abstract Strategies that enhance autophagy clearance of polyQ accumulation have become an attractive approach to revive neuronal cell viabilities. In the part I of the dissertation, a selected autophagic enhancer candidate was identified as a cell model for polyQ aggregation clearance. The polyQ diseases were caused by expansion of CAG trinucleotide repeats in the coding region of gene. After screening a series of triazole derivatives, a newly identified synthetic compound, 5,5′-(4,4′-(1,3-phenylenebis(oxy))-bis(methylene)-bis(1H-1,2,3-triazole-4,1-diyl))-bis(methylene)bis(3-(naphthalene-1-yl)-oxazolidin-2-one (OC-13), was shown capable of enhancing clearance of the aggregated polyQ in neuroblastoma cells. Human neuroblastoma cells SK-N-SH with ectopic expression of ΔCTBP-Q79-EGFP or Httex1-Q97-GFP mutant protein can be cleared of mutated aggregates without affecting cell viabilities. Treatment of OC-13 increased autophagosome formation and more than 50% the accumulated aggregates were eradicated as determined by fluorescence microscopy. Western blot showed that OC-13 converted LC3-I to LC3-II in the transfected cells and activated autophagy-mediated elimination of polyQ aggregation. The effects were repressed by autophagic inhibitors.. Autophagic enhancer is also a valid treatment strategy as cancer therapeutics. In the part II of the dissertation, another newly identified triazole derivative compound, 4-((5-benzyl-1H-1,2,4-triazol-3-yl)methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), was found inhibiting the growth of human NSCLC cells. BTO induced. 11.

(12) autophagic characteristics and inhibited cell growth as shown in MTT evaluation, colony forming assay and Western blot. The compound induced autophagosome and autolysosome formation. More experiments with Annexin V staining and Western blot showed that the drug induced apoptotic cell death that was related to autophagy activation. Furthermore, BTO suppressed the growth of xenograft tumors by activating autophagy-mediated apoptosis as shown in Western blot and fluorescence microscopy of tumor tissue specimen staining.. The triazole derivatives OC-13 and BTO can be of potential therapeutic values to treat human diseases.. Keywords: autophagic flux, polyglutamine, aggregates clearance, triazole, JNK pathway, neuronal disorders, human non-small-cell-lungcancer cells, apoptosis. 12.

(13) Chapter II 中文摘要本論文目的是鑑定三唑衍生物潛在用於治療神經退化疾病及非小細肺癌細胞的藥物。多麩醯胺酸造成的神經退化性疾病是因為神經細胞中基因座上出現過度 CAG 三核甘酸的重複序列所導致的疾病。利用增加細胞自噬清除多麩醯胺酸堆積，是一種可能的方式用來治療多麩醯胺酸相關神經疾病。在第一部分的論文中，利用表現多麩醯胺酸的細胞模式篩選一系列的候選藥物。從一系列三唑衍生物中篩選出化合物的藥物 OC-13,它可以透過增強自噬特性，清除外來質體衍生的蛋白質ΔC-TBP-Q79-EGFP 或 Httex1-Q97-GFP 造成多麩醯胺酸堆積，卻不會影響細胞活性。由螢光顯微鏡下觀察，表現擴增多麩醯胺酸的細胞經 OC-13 處理增加自噬小體，會清除超過５０％的蛋白質堆積。由西方墨點法分析顯示，OC-13 促進 LC3-I 轉換為 LC3-II 以增進自噬體形成，而自噬抑制劑能阻斷 OC-13 清除多麩醯胺酸的功能。論文第二部分利用增強細胞自噬主導的第二型計畫性細胞死亡可以用作於癌症的治療。透過細胞活性分析, colony forming assay, Annexin V 染色及西方墨點法分析，可以確認另一個三唑衍生物 BTO 會抑制人類非小細胞肺癌細胞的增生，並能隨著時間及濃度誘導自噬相關蛋白，並且促進凋亡，這都是透過自噬小體增加與. 13.

(14) 溶酶體的形成。利用 Annexin V 染色及西方墨點法分析可以顯示促進自噬的 BTO 造成肺癌細胞的凋亡。此外由腫瘤組織的西方墨點分析和螢光顯微鏡中顯示，BTO 可以抑制在小鼠模型中異種移植的 A549 腫瘤生長，並且誘導凋亡和自噬標記物的增生。. 這兩種三唑衍生物化合物可以是抑制人類疾病的一種潛在治療藥劑。. 關鍵字:細胞自噬，多麩醯胺酸，聚集物清除，三唑化合物，JNK 訊息傳遞，神經退化疾病，人類非小細胞肺癌細胞，細胞凋亡. 14.

(15) LIST OF ABBREVIATIONS. 3-MA AD ALS ANOVA Baf A1 BTO CDDP DAPI DMEM DMSO Dox ECL EDTA EGFP ELISA FACScalibur FBS GFP H&E H2DCFDA HD Httex1 IR JNK LC3 mp MS mTOR MTT NAC. 3-Methyladenine Alzheimer's disease Amyotrophic Lateral Sclerosis Analysis of variance Bafilomycin A1 4-((5-benzyl-1H-1,2,4-triazol-3-yl)-methyl)-7methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one Cisplatin 4',6-diamidino-2-phenylindole Dulbecco's Modified Eagle Medium Dimethyl sulfoxide Doxycycline Enhanced chemiluminescence Ethylenediaminetetraacetic acid Enhanced green fluorescent protein Enzyme-Linked ImmunoSorbent Assay Fluorescence-activated cell sorting Fetal bovine serum Green fluorescent protein Hematoxylin and eosin 2',7'-dichlorodihydrofluorescein diacetate Huntington’s disease Huntingtin exon 1 Infrared spectroscopy c-Jun N-terminal kinases Microtubule-associated protein 1A/1B-light chain 3 Melting point Mass spectroscopy mammalian target of Rapamycin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N-acetyl-L-cysteine. 15.

(16) NMR NSCLC OC-13. PBS PCNA PD PI3KC3 polyQ RIPA ROS SCLC SDS SMER SQSTM1 TBP TUNEL β-lap. Nuclear magnetic resonance non-small cell lung cancer (5,5′-(4,4′-(1,3-phenylene-bis(oxy))-bis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))-bis(methylene)-bis(3(naphthalene-1-yl)-oxazolidin-2-one) Phosphate-buffered saline Proliferating cell nuclear antigen Parkinson’s disease Class III phosphatidylinositol 3-kinase Polyglutamine Radioimmunoprecipitation assay Reactive oxygen species Small-cell carcinoma Sodium dodecyl sulfate Small-molecule enhancers of rapamycin Sequestosome-1 TATA-binding protein Terminal deoxynucleotidyl transferase dUTP nick end labeling assay β-Lapachone. 16.