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系統識別號 U0007-3007201110542100
論文名稱(中文) 利用反譯寡核苷酸微脂粒─G3139與自由基藥物之並用以提升癌細胞之細胞凋亡
論文名稱(英文) Using liposomal antisense oligonucleotide – G3139 and NO donor drugs to enhance apoptosis in cancer cell lines
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 99
學期 2
出版年 100
研究生(中文) 孫晟紘
研究生(英文) Chen-Hung Sun
學號 M301098019
學位類別 碩士
語文別 中文
口試日期 2011-06-17
論文頁數 77頁
口試委員 委員-林文貞
委員-廖嘉鴻
指導教授-邱士娟
中文關鍵字 反譯寡核苷    Bcl-2  微脂粒  SIN-1  一氧化氮(NO) 
英文關鍵字 Antisense oligonucleotide  Bcl-2  liposome  SIN-1  nitric oxide (NO) 
學科別分類
中文摘要 細胞凋亡是人體內一種引發細胞死亡的重要生理機制,主要是受到促進細胞凋亡和抑制細胞凋亡的兩種蛋白質所調控。而在細胞凋亡的途徑中,BCL-2是一個相當重要的抑制細胞凋亡的蛋白質,抑制BCL-2的表現可能能夠抑制癌細胞該途徑的表現。研究中所使用的G3139是一具有影響BCL-2 mRNA的反譯寡核苷酸。一氧化氮(Nitric oxide, NO)是一種重要的細胞信號分子,在包括細胞死亡等許多正常生理過程中扮演重要的角色。研究中選用了三種會釋放一氧化氮(NO donor)的藥物:S-Nitroso-N-acetylpenicillamine (SNAP)、S-Nitrosoglutathione (GSNO) 及 3-morpholinosydnonimine hydrochloride (SIN-1)來評估其殺死細胞的效果。本研究的目的是要開發一個包覆ODN(G3139)的微脂粒系統,可以有效的將G3139遞送到白血病細胞,並評估ODN相關的微脂粒劑型與NO donor藥物併用時對白血病細胞的細胞死亡之影響。
本研究是利用酒精注射法製備微脂粒,所使用的脂質組成為DC-Chol: Egg PC: PEG-DSPE = 22.5:76:1.5 (莫耳百分率),AsODN 與脂質的比例為 1:10 (重量比)。微脂粒的粒徑大小及表面電荷利用Zetasizer測量,並透過微脂粒粒徑大小之變化來評估其安定性。實驗中使用了三種白血病相關的細胞株:K562、NCI-H929及CCRF-CEM。給予細胞ODN相關之配方,並在預先選定的時間點進行RT-PCR的檢測,以觀察BCL-2的改變。在毒性試驗中,在投予藥物到細胞中的24或48小時後,利用MTS試劑來進行細胞毒性之評估。
包覆ODN之微脂粒的平均直徑為148.9±2.5 nm且有相當好的包覆率(85.5±5.3%),觀察六週後其粒徑大小並無明顯改變。在降低BCL-2表現的研究結果中,無論是給予未被包覆的ODN或微脂粒包覆的ODN,與對照組相比時,BCL-2 mRNA並無顯著的改變。經過GSNO、SIN-1或SNAP在K562細胞中單獨使用之細胞毒性試驗中,選定SIN-1作進一步的研究。研究中所使用之所有細胞,在有給予藥物的組別,當細胞培養的時間越長其細胞毒性就會越高。然而,不同的細胞株對藥物有不同的敏感性,其中K562細胞對SIN-1的敏感性是最差的。實驗中所有的細胞株,在給予SIN-1與包覆ODN的配方併用的組別相較於單獨使用SIN-1的組別均顯示較佳的細胞毒性,表示此兩物質併用時可以達到增強藥效的結果。使用三種ODN相關的配方中,SIN-1與ODN的併用有最低的細胞毒性的表現,而在實驗中所有的細胞株中,SIN-1併用空白之微脂粒與包覆ODN之微脂粒的細胞毒性有相似的表現。至於造成其增強作用的機制為何,則需要更進一步的研究才能得知。
英文摘要 Apoptosis is an important physiological mechanism in programmed cell death in human. It is a process majorly regulated by two groups of proteins, pro-apoptotic and anti-apoptotic proteins. Bcl-2 is one of the most important anti-apoptotic proteins in apoptosis pathway. The downregulation of Bcl-2 may be able to inhibit this pathway in cancer cells. G3139, an antisense oligonucleotide specific targeting to Bcl-2 mRNA, was used in this study. NO is an important cell signaling molecule in many biological processes, including cell death. Three molecules that can release NO (NO donor drugs), S-Nitroso-N-acetylpenicillamine (SNAP)、S-Nitrosoglutathione (GSNO) and 3-morpholinosydnonimine hydrochloride (SIN-1) were evaluated in the current study. The purpose of this study is to develop a ODN (G3139)-containing liposome system that can efficiently deliver G3139 to leukemia cells and to evaluate the combination effect of cell death by this ODN-related liposomal formulation and NO donor drugs in leukemia cells.
ODN-containing liposomes were prepared by ethanol injection method. Liposomes composed of DC-Chol/egg PC/PEG-DSPE (22.5:76:1.5, mole/mole) were loaded with G3139 with ODN-to-total lipid ratio of 1:10 (wt ratio). Particle size and zeta potential of liposomes were measured by Zetasizer. The stability of ODN-containing liposomes was evaluated by monitoring the particle size change. Three leukemia-related cell lines, K562, NCI-H929, and CCRF-CEM were used in the current study. Cells were treated with ODN-related formulations for up to 48 hr and were collected in pre-selected time point for RT-PCR assay to monitor the Bcl-2 downregulation effect. In cytotoxicity studies, cells were treated for 24 or 48 hr and were evaluated by MTS assay.
The ODN-containing liposomes had mean diameter of 148.9±2.5 nm with great encapsulation efficiency of ODN (85.5±5.3%). No significant particle size change of ODN-containing liposomes was observed for up to 6 weeks. The results in BCl-2 downregulation studies showed that there was no significant Bcl-2 mRNA change in these groups (free ODN or ODN-liposomes) comparing with control group. After testing the cytotoxic effect of GSNO, SIN-1, or SNAP alone in K562 cells, SIN-1 was chosen for further studies. In all cell lines used in the study, cells demonstrated significantly higher cytotoxic effect in all treatment groups as incubation time period increased. However, the sensitivity of drugs in different cell lines was different, while K562 exhibited the lowest sensitivity of SIN-1. Cells treated with SIN-1 combined with ODN-related formulations had significantly higher cytotoxic effect than SIN-1 alone in all cell lines. Among three ODN-related formulations used, SIN-1 combined with free ODN showed lowest cytotoxic effect in all three cell lines and empty liposomes and ODN-liposomes exhibited similar cytoxic effect. Further studies are warranted to explore the underlying mechanism of the combination effect.
論文目次 中文摘要-------------------------------------------------2
英文摘要-------------------------------------------------4
圖目錄---------------------------------------------------9
緒論-----------------------------------------------------9
一、 文獻探討--------------------------------------10
(一) 細胞凋亡-----------------------------------10
(二) BCL-2蛋白家族-----------------------------12
(三) 反譯寡核苷酸-------------------------------14
(四) G3139--------------------------------------16
(五) 傳遞系統-----------------------------------18
(六) Nitric Oxide--------------------------------21
二、 研究動機--------------------------------------24
實驗材料及方法-------------------------------------------25
一、 實驗細胞及材料--------------------------------25
(一) 實驗細胞-----------------------------------25
(二) 實驗藥品-----------------------------------26
二、 實驗方法--------------------------------------26
(一) 細胞培養-----------------------------------29
(二) Total RNA製備------------------------------31
(三) 反轉錄聚合脢連鎖反應-----------------------32
(四) 製備微脂粒---------------------------------34
(五) 微脂粒包覆能力試驗-------------------------35
(六) 細胞存活率試驗-----------------------------36
(七) Nitric Oxide 的釋放試驗---------------------37
(八) 統計分析方法-------------------------------38
實驗結果-------------------------------------------------39
一、 微脂粒之物理化學性質
(一) 微脂粒包覆率
(二) 粒徑大小試驗
(三) 微脂粒的表面電荷
(四) 微脂粒安定性試驗
二、 不同劑型之ODN對BCL-2基因的RT-PCR試驗
三、 細胞存活率試驗
(一) DMSO之劑量影響曲線
(二) 不同的NO donor藥物在K562之劑量影響曲線
(三) SIN-1與不同的formulations之併用之細胞存活率試驗
1. K562:SIN-1 + different formulations
2. NCI-H929:SIN-1 + different formulations
3. CCRF-CEM:SIN-1 + different formulations
(四) 空白微脂粒在癌細胞之劑量影響曲線
(五) Nitric Oxide的速放速率試驗
(六) SIN-1與空白微脂粒之併用在癌細胞之細胞存活率試驗
討論-----------------------------------------------------68
結論-----------------------------------------------------72
參考文獻-------------------------------------------------73
參考文獻 1. Lessene, G., P.E. Czabotar, and P.M. Colman, BCL-2 family antagonists for cancer therapy. Nature Reviews Drug Discovery, 2008. 7(12): p. 989-1000.
2. Yip, K.W. and J.C. Reed, Bcl-2 family proteins and cancer. Oncogene, 2008. 27(50): p. 6398-6406.
3. Kerr, J.F., A.H. Wyllie, and A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 1972. 26(4): p. 239-257.
4. Nagata, S., R. Hanayama, and K. Kawane, Autoimmunity and the Clearance of Dead Cells. Cell, 2010. 140(5): p. 619-630.
5. Ow, Y.L.P., et al., Cytochrome c: Functions beyond respiration. Nature Reviews Molecular Cell Biology, 2008. 9(7): p. 532-542.
6. Nagata, S., Apoptosis by death factor. Cell, 1997. 88(3): p. 355-365.
7. Strasser, A., P.J. Jost, and S. Nagata, The Many Roles of FAS Receptor Signaling in the Immune System. Immunity, 2009. 30(2): p. 180-192.
8. Cory, S., D.C.S. Huang, and J.M. Adams, The Bcl-2 family: Roles in cell survival and oncogenesis. Oncogene, 2003. 22(53 REV. ISS. 7): p. 8590-8607.
9. Levine, B. and G. Kroemer, Autophagy in the Pathogenesis of Disease. Cell, 2008. 132(1): p. 27-42.
10. Reed, J.C., Bcl-2-family proteins and hematologic malignancies: history and future prospects. Blood, 2008. 111(7): p. 3322-3330.
11. Reed, J.C., Proapoptotic multidomain Bcl-2/Bax-family proteins: Mechanisms, physiological roles, and therapeutic opportunities. Cell Death and Differentiation, 2006. 13(8): p. 1378-1386.
12. Chiu, S.-J., et al., Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes. Journal of Controlled Release, 2006. 112(2): p. 199-207.
13. Dias, N. and C.A. Stein, Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides. European Journal of Pharmaceutics and Biopharmaceutics, 2002. 54(3): p. 263-269.
14. Pan, X., et al., Antitumor activity of G3139 lipid nanoparticles (LNPs). Molecular Pharmaceutics, 2009. 6(1): p. 211-220.
15. Paterson, B.M., B.E. Roberts, and E.L. Kuff, Structural gene identification and mapping by DNA.mRNA hybrid-arrested cell-free translation. Proceedings of the National Academy of Sciences of the United States of America, 1977. 74(10): p. 4370-4374.
16. Rayburn, E.R. and R. Zhang, Antisense, RNAi, and gene silencing strategies for therapy: Mission possible or impossible? Drug Discovery Today, 2008. 13(11-12): p. 513-521.
17. Baker, B.F. and B.P. Monia, Novel mechanisms for antisense-mediated regulation of gene expression. Biochimica et Biophysica Acta - Gene Structure and Expression, 1999. 1489(1): p. 3-18.
18. Yao, Y., et al., Antisense makes sense in engineered regenerative medicine. Pharmaceutical Research, 2009. 26(2): p. 263-275.
19. Askari, F.K. and W.M. McDonnell, Molecular medicine: Antisense-oligonucleotide therapy. New England Journal of Medicine, 1996. 334(5): p. 316-318.
20. Koh, C.G., et al., Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing. Journal of Controlled Release, 2010. 141(1): p. 62-69.
21. Saul, J.M., et al., Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression. Biomaterials, 2007. 28(31): p. 4705-4716.
22. Pirollo, K.F., et al., Antisense therapeutics: From theory to clinical practice. Pharmacology and Therapeutics, 2003. 99(1): p. 55-77.
23. Milhavet, O., D.S. Gary, and M.P. Mattson, RNA Interference in Biology and Medicine. Pharmacological Reviews, 2003. 55(4): p. 629-648.
24. Kurreck, J., Antisense technologies: Improvement through novel chemical modifications. European Journal of Biochemistry, 2003. 270(8): p. 1628-1644.
25. Richardson, D.R., et al., Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents. Biochimica et Biophysica Acta - General Subjects, 2009. 1790(7): p. 702-717.
26. Frankel, S.R., Oblimersen sodium (G3139 Bcl-2 antisense oligonucleotide) therapy in Waldenstrom's macroglobulinemia: A targeted approach to enhance apoptosis. Seminars in Oncology, 2003. 30(2): p. 300-304.
27. Moulder, S.L., et al., Phase I/II study of G3139 (Bcl-2 antisense oligonucleotide) in combination with doxorubicin and docetaxel in breast cancer. Clinical Cancer Research, 2008. 14(23): p. 7909-7916.
28. Stein, C.A., et al., G3139, an Anti-Bcl-2 Antisense Oligomer That Binds Heparin-Binding Growth Factors and Collagen I, Alters In vitro Endothelial Cell Growth and Tubular Morphogenesis. Clinical Cancer Research, 2009. 15(8): p. 2797-2807.
29. Liu, G., et al., A phase i pharmacokinetic and pharmacodynamic correlative study of the antisense Bcl-2 oligonucleotide G3139, in combination with carboplatin and paclitaxel, in patients with advanced solid tumors. Clinical Cancer Research, 2008. 14(9): p. 2732-2739.
30. Chen, A.M., et al., Co-delivery of Doxorubicin and Bcl-2 siRNA by Mesoporous Silica Nanoparticles Enhances the Efficacy of Chemotherapy in Multidrug-Resistant Cancer Cells. Small, 2009. 5(23): p. 2673-2677.
31. Bedikian, A.Y., et al., Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: The oblimersen melanoma study group. Journal of Clinical Oncology, 2006. 24(29): p. 4738-4745.
32. Li, S.D. and L. Huang, Non-viral is superior to viral gene delivery. Journal of Controlled Release, 2007. 123(3): p. 181-183.
33. Karmali, P.P. and A. Chaudhuri, Cationic liposomes as non-viral carriers of gene medicines: Resolved issues, open questions, and future promises. Medicinal Research Reviews, 2007. 27(5): p. 696-722.
34. Walther, W. and U. Stein, Viral vectors for gene transfer: A review of their use in the treatment of human diseases. Drugs, 2000. 60(2): p. 249-271.
35. Al-Dosari, M.S. and X. Gao, Nonviral gene delivery: Principle, limitations, and recent Progress. AAPS Journal, 2009. 11(4): p. 671-681.
36. Sokolova, V. and M. Epple, Inorganic nanoparticles as carriers of nucleic acids into cells. Angewandte Chemie - International Edition, 2008. 47(8): p. 1382-1395.
37. Cai, X., S. Conley, and M. Naash, Nanoparticle applications in ocular gene therapy. Vision Research, 2008. 48(3): p. 319-324.
38. Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(16): p. 7297-7301.
39. Morille, M., et al., Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials, 2008. 29(24-25): p. 3477-3496.
40. Juliano, R., et al., Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Research, 2008. 36(12): p. 4158-4171.
41. Hwang, S.J. and M.E. Davis, Cationic polymers for gene delivery: Designs for overcoming barriers to systemic administration. Current Opinion in Molecular Therapeutics, 2001. 3(2): p. 183-191.
42. Glodde, M., S.R. Sirsi, and G.J. Lutz, Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides. Biomacromolecules, 2006. 7(1): p. 347-356.
43. Felgner, P.L., et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences of the United States of America, 1987. 84(21): p. 7413-7417.
44. Durcan, N., C. Murphy, and S.A. Cryan, Inhalable siRNA: Potential as a therapeutic agent in the lungs. Molecular Pharmaceutics, 2008. 5(4): p. 559-566.
45. Farjo, R., et al., Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS ONE, 2006. 1(1).
46. Tranchant, I., et al., Physicochemical optimisation of plasmid delivery by cationic lipids. Journal of Gene Medicine, 2004. 6(SUPPL. 1): p. S24-S35.
47. Drummond, D.C., et al., Development of a highly stable and targetable nanoliposomal formulation of topotecan. Journal of Controlled Release, 2010. 141(1): p. 13-21.
48. Gupta, U. and N.K. Jain, Non-polymeric nano-carriers in HIV/AIDS drug delivery and targeting. Advanced Drug Delivery Reviews, 2010. 62(4-5): p. 478-490.
49. Klegerman, M.E., et al., Liposomal modular complexes for simultaneous targeted delivery of bioactive gases and therapeutics. Journal of Controlled Release, 2010. 142(3): p. 326-331.
50. Minko, T., HPMA copolymers for modulating cellular signaling and overcoming multidrug resistance☆. Advanced Drug Delivery Reviews, 2010. 62(2): p. 192-202.
51. Schrammel, A., et al., Activation of soluble guanylyl cyclase by the nitrovasodilator 3- morpholinosydnonimine involves formation of S-nitrosoglutathione. Molecular Pharmacology, 1998. 54(1): p. 207-212.
52. Kang, J.L., K. Lee, and V. Castranova, Nitric oxide up-regulates DNA-binding activity of nuclear factor-κB in macrophages stimulated with silica and inflammatory stimulants. Molecular and Cellular Biochemistry, 2000. 215(1-2): p. 1-9.
53. Scarpato, R., et al., Cytotoxicity and genotoxicity studies of two free-radical generators (AAPH and SIN-1) in human microvascular endothelial cells (HMEC-1) and human peripheral lymphocytes. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2011. 722(1): p. 69-77.
54. Ascenzi, P., et al., Inhibition of cysteine protease activity by NO-donors. Current Protein and Peptide Science, 2001. 2(2): p. 137-153.
55. Shaffer, J.E., et al., Lack of tolerance to a 24-hour infusion of S-nitroso N-acetylpenicillamine (SNAP) in conscious rabbits. Journal of Pharmacology and Experimental Therapeutics, 1992. 260(1): p. 286-293.
56. Hogg, N., et al., Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochemical Journal, 1992. 281(2): p. 419-424.


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