(3.236.214.19) 您好!臺灣時間:2021/05/06 22:10
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:張耀仁
研究生(外文):Yao- Jen Chang
論文名稱:分析植物對苯二酚衍生物HQ17(3)對帶有費城染色體之急性淋巴性白血病細胞株SUP-B15的抑制作用
論文名稱(外文):Inhibitory effects of the botanical alkyl hydroquinone derivative HQ17(3)on acute lymphoblastic leukemia SUP-B15 cells harboring Philadelphia chromosome
指導教授:胡忠怡
口試委員:林淑萍林亮音張雅雯楊雅倩
口試日期:2013-06-28
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:95
中文關鍵詞:急性淋巴性白血病SUP-B15HQ17(3)細胞自噬活性氧族群
外文關鍵詞:acute lymphoblastic leukemia (ALL)SUP-B15HQ17(3)autophagyreactive oxygen species (ROS)
相關次數:
  • 被引用被引用:0
  • 點閱點閱:225
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
人類第9 號染色體與第22 號染色體發生轉位之後產生費城染色體(Philadelphia chromosome),因而產生具有致癌性的融合蛋白BCR-ABL。BCR-ABL 為持續活化的酪胺酸激酶(tyrosine kinase),會啟動下游許多關於細胞增生、存活、以及自我更新的訊號傳遞,而導致細胞惡性轉型。分子量為190 kD 的BCR-ABL 融合蛋白多發現於急性淋巴性白血病(ALL) (Ph+ ALL),其疾病惡性度高,預後不佳;現階段以最高強度化學治療搭配tyrosine kinase inhibitors (TKI,如: Imatinib, Dasatinib)抑制BCR-ABL的活性,雖可使病人獲得暫時性緩解,但是仍有高比例病人血癌復發,並且發生抗藥性。因此,發展有別於化療的藥物或與tyrosine kinase inhibitor 合併搭配使用,是治療Ph+ ALL 的重要課題。10¢(Z),13¢(E),15¢(E)-heptadecatrienyl
hydroquinone (HQ17(3))是萃取自漆樹的天然小分子,對於多種腫瘤細胞具有毒殺能力,但對於人類正常周邊血液單核細胞以及實驗鼠無明顯毒性,HQ17(3)在腫瘤細胞中具有抑制DNA 拓樸異構酶II (DNA topoisomerase II)以及誘導產生reactiveoxygen species (ROS)能力,並可在肝癌細胞株中引起DNA 損傷及apoptotic cell death。我們在前驅測試中發現以低濃度HQ17(3)處理帶有BCR-ABL 的ALL 細胞株-SUP-B15 24 小時即有顯著毒殺作用。
本研究結果顯示HQ17(3)會誘導SUP-B15 細胞活性氧(ROS)上升、酸性胞器出
現、粒線體膜電位喪失、染色體斷裂及細胞自噬標記LC3-II 產生。抗氧化劑
(Antioxidantss, ROS scavenger)穀胱苷肽(GSH)與維生素C 可以減緩HQ17(3)所誘導的ROS 產生、減少粒線體膜電位損傷並降低細胞死亡。親脂性排鐵劑desferrioxamine mesylate (DFO)可阻止酸性胞器產生,並阻斷HQ17(3)所引起的粒線體損傷及細胞死亡;加入抑制細胞自噬抑制劑3methyl adenine (3-MA)與氯奎寧(Chloroquine)亦可以減少HQ17(3)誘發之細胞死亡。綜合本研究的實驗結果顯示HQ17(3)誘導ROS 啟動細胞自噬可能是造成SUP-B15 細胞死亡重要原因,並且此過程高度依賴鐵存在。因此,本研究結果指出,若可誘導癌細胞產生細胞自噬將有潛力發展輔助治療帶費城染色體急性淋巴性白血病的策略。

Reciprocal t(9;22); BCR-ABL translocation gives rise to Philadelphia(Ph)chromosome and results in production of chimeric BCR-ABL fusion protein with constitutively active tyrosine kinase activity. The BCR-ABL protein activates a number of signaling pathways which promote cell proliferation, survival and self-renewal. Ph+ acute lymphoblastic leukemia (ALL) with 190kD BCR-ABL fusion protein presents very poor clinical outcomes. Although tyrosine kinase inhibitor (TKI) combined with multi-agent chemotherapy help to acquire a complete remission temporarily, still a high proportion of patients have leukemic relapse and develop drug resistance. Therefore,
developing drug(s) to act in different ways as in chemotherapies, or to be used in combination with the TKI in treatment of Ph+ ALL, is an alternative way to help the
patients affected by this very high risk disease. 10¢(Z),13¢(E),15¢(E)-heptadecatrienyl hydroquinone (HQ17(3)) is a small natural molecule extracted from the R. succedanea.
DNA topoisomerase IIα inhibition and oxidative stress were found to account for selective cytotoxicity caused by HQ17(3) in various types of tumor cells. In the preliminary data, we found that HQ17(3) has significant cytotoxic effect in a p190 BCR-ABL Ph+ ALL cell line, SUP-B15, within 24 hours in micromolar concentration.
We found in SUP-B15 cells, HQ17(3) induced reactive oxygen species (ROS) and acidic vesicle formation, mitochondrial membrane potential disturbance, chromosome breakage, and emergence of an autophagy marker, cleaved LC3-II. ROS scavengers(Glutathione, vitamin C) attenuated HQ17(3)-induced cell injury. Lysosomotropic iron chelator, desferrioxamine mesylate (DFO) abolished HQ17(3)-induced acidic vesicles formation, mitochondrial membrane potential loss and cell death. Inhibitors for autophagy (3-methyl adenine, chloroquine) partially rescued cells from
HQ17(3)-induced death. These results indicated that HQ17(3) may induce ROS production, and subsequently lead to autophagic cell death. In conclusion, HQ17(3) displayed a significant anti-leukemic activity in Ph+ ALL (SUP-B15) cells by ROS production and lysosomal iron-dependent events that contribute to autophagic cell death. These results suggest that agents selectively induce ROS in leukemic cells might induce autophagic cell death, and would potentially augment the treatment for Ph+ ALL.

致謝I
中文摘要II
AbstractIV
縮寫表VI
第一章緒論1
第一節急性淋巴性白血病1
1.1 急性淋巴性白血病的症狀1
1.2 急性淋巴性白血病1
1.3 急性淋巴性白血病的診斷2
1.4 急性淋巴性白血病的治療2
1.5 急性淋巴性白血病的預後因子4
1.6 費城染色體陽性急性淋巴性白血病簡介4
1.7 費城染色體5
1.8 費城染色體陽性急性淋巴性白血病治療5
1.9 以IM 為基底的治療在Ph+ ALL 所面臨的問題6
第二節對苯二酚衍生物HQ17(3)簡介7
2.1 對苯二酚的代謝7
2.2 對苯二酚相關文獻探討7
2.3 DNA 拓樸異構酶(DNA topoisomerases II, Topo II)8
2.4 對苯二酚衍生物-HQ17(3)相關文獻探討8
第三節活性氧分子reactive oxygen species9
3.1 活性氧分子reactive oxygen species9
3.2 ROS 與細胞死亡11
第四節計畫性細胞死亡(Programmed cell death)12
4.1 細胞死亡定義12
4.2 細胞凋亡(Apoptosis, type I cell death)12
4.3 計畫性細胞壞死(Necroptosis)14
4.4 由Lysosome(溶小體)主導的細胞死亡方式15
4.4.1 Lysosome 的介紹15
4.4.2 Lysosomal membrane permeabilization (LMP)15
4.4.3 LMP 的偵測16
4.5 細胞自噬(Autophagy, type II cell death)17
4.5.1 autophagic cell death17
4.5.2 autophagy 的偵測19
第二章研究目的與實驗設計20
第一節研究目的20
1.1 研究目的20
1.2 研究動機20
第二節實驗設計21
第三章材料與方法22
第一節實驗材料22
1.1 細胞株22
1.2 試藥/劑、抗體、儀器、耗材清單22
1.3 各式溶液及其配方26
1.3.1 細胞培養、繼代26
1.3.2 流式細胞儀相關實驗26
1.3.3 萃取細胞蛋白質27
1.3.4 鈉十二烷基硫酸鹽聚丙烯胺凝膠電泳與膠體轉漬27
1.3.5 西方墨點法30
1.3.6 細胞存活/活性分析31
1.3.7 Sub-G1 分析 (chromosome fragmentation 分析)32
第二節實驗方法 32
2.1 解凍細胞、細胞培養及細胞計數32
2.2 細胞活性測試及藥物IC50 33
2.2.1 trypan blue exclusion assay33
2.2.2 ACP assay 33
2.3 PS 磷脂質外翻特徵與細胞膜完整性分析 (Annexin V/PI stain)33
2.4 細胞粒線體膜電位的測定(DiOC6(3) stain)34
2.5 Chromosome DNA fragmentation assay (sub-G1 fraction in DNA content analysis)35
2.6 細胞內caspase 活性分析(西方墨點法 35
2.6.1 蛋白質萃取 35
2.6.2 蛋白質定量、稀釋與電泳樣品準備 36
2.6.3 鈉十二烷基硫酸鹽聚丙烯胺凝膠電泳與膠體轉漬 (SDS-PAGE and
blotting)36
2.6.4 阻斷非特異性結合與免疫染色36
2.7 螢光染色 37
第四章結果38
第一節 HQ17(3)對於SUP-B15 細胞具有毒性 38
1.1 SUP-B15 對於imatinib 有抗性38
1.2 HQ17(3)對於SUP-B15 細胞具有毒性 38
1.3 HQ17(3)對於正常血球細胞不具毒殺能力39
第二節 HQ17(3)處理細胞後,SUP-B15 出現死亡特徵 39
2.1 HQ17(3)促使SUP-B15 細胞膜脂質外翻、細胞膜受損39
2.2 HQ17(3)處理細胞後,SUP-B15 細胞出現粒線體膜電位的喪失 40
第三節 HQ17(3)處理細胞後,引起ROS 相關路徑40
3.1 HQ17(3)誘導SUP-B15 細胞產生ROS40
3.2 抗氧化劑可以減少HQ17(3)所造成的細胞損傷 41
第四節 HQ17(3)引起caspase-independent cell death42
4.1 HQ17(3)僅誘導微量的caspase 3活化 42
4.2 pan-caspase inhibitor 無法挽救細胞死亡 42
第五節 HQ17(3)與Necroptosis 42
5.1 Necrostatin-1 無法挽救細胞死亡42
5.2 同時處理z-VAD 與Nec-1 無法挽救細胞死亡43
第六節 HQ17(3)可誘導酸性胞器表現 43
6.1 HQ17(3)處理細胞後酸性胞器增多 43
6.2 HQ17(3)可造成iron-dependent 細胞死亡 44
6.3 鐵螯合劑可減少酸性胞器產生 44
6.4 Lysosome 內酸性水解酶非導致SUP-B15 死亡原因45
6.5 HQ17(3)誘導細胞產生autophagy 45
6.6 抑制autophagy 可挽救部分細胞死亡46
6.7 DFO 抑制autophagy 產生46
第五章討論 48
第六章參考文獻 53
圖與表 59
附錄 82

1. 中華民國兒童癌症基金會: 兒童白血病, 2007
2. 施麗雲: 急性白血病衛教手冊
3. A.V. Hoffbrand JEPaPAHM: Essential Haematology fourth edition
4. 行政院衛生署國民健康局: 中華民國 97 年癌症登記報告, 2010
5. Thomas DA: Philadelphia chromosome positive acute lymphocytic leukemia: a new era of challenges, Hematology /the Education Program of the American Society of Hematology American Society of Hematology Education Program 2007, 435-443
6. Group TPO: Protocol: Taiwan Pediatric Oncology Group (TPOG)-ALL 2002.
7. Pui CH, Evans WE: Treatment of acute lymphoblastic leukemia, N Engl J Med 2006, 354:166-178
8. Pui CH, Relling MV, Downing JR: Acute lymphoblastic leukemia, N Engl J Med 2004, 350:1535-1548
9. Bentz M, Cabot G, Moos M, Speicher MR, Ganser A, Lichter P, Dohner H: Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization, Blood 1994, 83:1922-1928
10. Carpiuc KT, Stephens JM, Botteman MF, Feng W, Hay JW: A review of the clinical and economic outcomes of imatinib in Philadelphia chromosome-positive acute lymphoblastic leukemia, Expert opinion on pharmacotherapy 2007, 8:2775-2787
11. Fielding AK: Current treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia, Hematology / the Education Program of the American Society of Hematology American Society of Hematology Education Program 2011, 2011:231-237
12. Sawyers CL: Chronic myeloid leukemia, N Engl J Med 1999, 340:1330-1340
13. Mizuta S, Matsuo K, Yagasaki F, Yujiri T, Hatta Y, Kimura Y, Ueda Y, Kanamori H, Usui N, Akiyama H, Miyazaki Y, Ohtake S, Atsuta Y, Sakamaki H, Kawa K, Morishima Y, Ohnishi K, Naoe T, Ohno R: Pre-transplant imatinib-based therapy improves the outcome of allogeneic hematopoietic stem cell transplantation for BCR-ABL-positive acute lymphoblastic leukemia, Leukemia 2011, 25:41-47
14. Ko BS, Tang JL, Tsai W, Chen YC, Wang CH, Sheng MC, Lin DT, Lin KH, Tien HF: Philadelphia chromosome-positive acute lymphoblastic leukemia in Taiwan, Ann Hematol 2001, 80:510-515
15. Fielding AK: How I treat Philadelphia chromosome-positive acute lymphoblastic leukemia, Blood 2010, 116:3409-3417
16. Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, White D, Hughes TP, Le Beau MM, Pui CH, Relling MV, Shurtleff SA, Downing JR: BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros, Nature 2008, 453:110-114
17. Regev L, Wu M, Zlotolow R, Brautbar N: Hydroquinone, a benzene metabolite, and leukemia: a case report and review of the literature, Toxicol Ind Health 2012, 28:64-73
18. McGregor D: Hydroquinone: an evaluation of the human risks from its carcinogenic and mutagenic properties, Crit Rev Toxicol 2007, 37:887-914
19. Siew EL, Chan KM, Williams GT, Ross D, Inayat-Hussain SH: Protection of hydroquinone-induced apoptosis by downregulation of Fau is mediated by NQO1, Free Radic Biol Med 2012, 53:1616-1624
20. Inayat-Hussain SH, Ross D: Intrinsic pathway of hydroquinone induced apoptosis occurs via both caspase-dependent and caspase-independent mechanisms, Chem Res Toxicol 2005, 18:420-427
21. Lindsey RH, Jr., Bender RP, Osheroff N: Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison, Chem Res Toxicol 2005, 18:761-770
22. Wu PL, Lin SB, Huang CP, Chiou RY: Antioxidative and cytotoxic compounds extracted from the sap of Rhus succedanea, J Nat Prod 2002, 65:1719-1721
23. Huang CP, Fang WH, Lin LI, Chiou RY, Kan LS, Chi NH, Chen YR, Lin TY, Lin SB: Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning, Toxicol Appl Pharmacol 2008, 227:331-338
24. Lin TY, Huang CP, Au LC, Chang YW, Hu CY, Lin SB: A cysteine-reactive alkyl hydroquinone modifies topoisomerase IIalpha, enhances DNA breakage, and induces apoptosis in cancer cells, Chem Res Toxicol 2012, 25:2340-2351
25. Trachootham D, Alexandre J, Huang P: Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?, Nat Rev Drug Discov 2009, 8:579-591
26. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease, Int J Biochem Cell Biol 2007, 39:44-84
27. Fleury C, Mignotte B, Vayssiere JL: Mitochondrial reactive oxygen species in cell death signaling, Biochimie 2002, 84:131-141
28. Sidoti-de Fraisse C, Rincheval V, Risler Y, Mignotte B, Vayssiere JL: TNF-alpha activates at least two apoptotic signaling cascades, Oncogene 1998, 17:1639-1651
29. Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, Guerin CJ, Brunk UT, Declercq W, Vandenabeele P: Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features, Cell Death Differ 2010, 17:922-930
30. Boya P, Kroemer G: Lysosomal membrane permeabilization in cell death,Oncogene 2008, 27:6434-6451
31. Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH, Lenardo M: Autophagic programmed cell death by selective catalase degradation, Proc Natl Acad Sci U S A 2006, 103:4952-4957
32. Kreuzaler P, Watson CJ: Killing a cancer: what are the alternatives?, Nat Rev Cancer 2012, 12:411-424
33. Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, Gottlieb E, Green DR, Hengartner MO, Kepp O, Knight RA, Kumar S, Lipton SA, Lu X, Madeo F, Malorni W, Mehlen P, Nunez G, Peter ME, Piacentini M, Rubinsztein DC, Shi Y, Simon HU, Vandenabeele P, White E, Yuan J, Zhivotovsky B, Melino G, Kroemer G: Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012, Cell Death Differ 2012, 19:107-120
34. Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G: Molecular mechanisms of necroptosis: an ordered cellular explosion, Nature reviews Molecular cell biology 2010, 11:700-714
35. Chaitanya GV, Steven AJ, Babu PP: PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration, Cell Commun Signal 2010, 8:31
36. Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack G, Alnemri ES: In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains, Proc Natl Acad Sci U S A 1996, 93:7464-7469
37. Taylor RC, Cullen SP, Martin SJ: Apoptosis: controlled demolition at the cellular level, Nature reviews Molecular cell biology 2008, 9:231-241
38. Kroemer G, Martin SJ: Caspase-independent cell death, Nat Med 2005, 11:725-730
39. Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury, Nat Chem Biol 2005, 1:112-119
40. Feoktistova M, Geserick P, Kellert B, Dimitrova DP, Langlais C, Hupe M, Cain K, MacFarlane M, Hacker G, Leverkus M: cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms, Mol Cell 2011, 43:449-463
41. Kroemer G, Jaattela M: Lysosomes and autophagy in cell death control, Nat Rev Cancer 2005, 5:886-897
42. Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ: Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent, Am J Physiol Gastrointest Liver Physiol 2002, 283:G947-956
43. Levine B, Kroemer G: Autophagy in the pathogenesis of disease, Cell 2008,
132:27-42
44. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G: Self-eating and self-killing:
crosstalk between autophagy and apoptosis, Nature reviews Molecular cell biology 2007, 8:741-752
45. Bellodi C, Lidonnici MR, Hamilton A, Helgason GV, Soliera AR, Ronchetti M, Galavotti S, Young KW, Selmi T, Yacobi R, Van Etten RA, Donato N, Hunter A, Dinsdale D, Tirro E, Vigneri P, Nicotera P, Dyer MJ, Holyoake T, Salomoni P, Calabretta B: Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells, J Clin Invest 2009, 119:1109-1123
46. Altman BJ, Jacobs SR, Mason EF, Michalek RD, MacIntyre AN, Coloff JL, Ilkayeva O, Jia W, He YW, Rathmell JC: Autophagy is essential to suppress cell stressand to allow BCR-Abl-mediated leukemogenesis, Oncogene 2011, 30:1855-1867
47. Kao Y-W: The effects of botanical alkyl hydroquinone derivative HQ17(3) on acute lymphoblastic leukemia cell line,RS4;11 harboring t(4;11) chromosome translocation, 2011,
48. Sukhai MA, Prabha S, Hurren R, Rutledge AC, Lee AY, Sriskanthadevan S, Sun H, Wang X, Skrtic M, Seneviratne A, Cusimano M, Jhas B, Gronda M, MacLean N, Cho EE, Spagnuolo PA, Sharmeen S, Gebbia M, Urbanus M, Eppert K, Dissanayake D, Jonet A, Dassonville-Klimpt A, Li X, Datti A, Ohashi PS, Wrana J, Rogers I, Sonnet P, Ellis WY, Corey SJ, Eaves C, Minden MD, Wang JC, Dick JE, Nislow C, Giaever G, Schimmer AD: Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors, J Clin Invest 2013, 123:315-328
49. Devireddy LR, Gazin C, Zhu X, Green MR: A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake, Cell 2005, 123:1293-1305
50. de Bruin EC, Meersma D, de Wilde J, den Otter I, Schipper EM, Medema JP, Peltenburg LT: A serine protease is involved in the initiation of DNA damage-induced apoptosis, Cell Death Differ 2003, 10:1204-1212
51. Laane E, Tamm KP, Buentke E, Ito K, Kharaziha P, Oscarsson J, Corcoran M, Bjorklund AC, Hultenby K, Lundin J, Heyman M, Soderhall S, Mazur J, Porwit A, Pandolfi PP, Zhivotovsky B, Panaretakis T, Grander D: Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy, Cell Death Differ 2009, 16:1018-1029
52. Castino R, Fiorentino I, Cagnin M, Giovia A, Isidoro C: Chelation of lysosomal iron protects dopaminergic SH-SY5Y neuroblastoma cells from hydrogen peroxide toxicity by precluding autophagy and Akt dephosphorylation, Toxicol Sci 2011, 123:523-541

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔