臺灣博碩士論文加值系統

在很多癌症組織或細胞中發現組蛋白乙醯酶(histone acetylases；HATs)
和組蛋白去乙醯酶(histone deacetylases；HDACs)處於不平衡的狀態。
組蛋白去乙醯酶在腫瘤的形成和血管新生扮演關鍵的角色。近年來，
組蛋白去乙醯酶抑制劑快速的開發，它會反轉癌症中不正常表觀遺傳
的改變，對於抗癌是ㄧ個非常有前景的發展。Psammaplin A (PsA)是
ㄧ個天然的溴化酪胺酸衍生物，1987 年首次從Psammaplysilla sponge
這種海綿中萃取出來。過去的文獻指出PsA 會抑制組蛋白去乙醯酶和
DNA 甲基轉移酶，如同腫瘤抑制基因的表觀遺傳修飾，因此為一種
新興的組蛋白去乙醯酶抑制劑。由於還未在人類子宮頸癌細胞上研究
過，其分子機轉也不太清楚，因此我們主要想探討PsA 對人類子宮頸
癌細胞的抗癌效力。結果顯示，PsA 會誘發p21 表現量使細胞週期休
止在G1 期而抑制人類子宮頸癌細胞HeLa 的增殖，並且誘發凋亡性
的細胞死亡。並且PsA 會抑制Mdm2 蛋白的表現量，並且增加它的
泛素化程度，因而降低它與p53 結合的能力，使p53 蛋白泛素化的程
度降低，而影響p53 蛋白的穩定性，延長p53 蛋白的半衰期，而使得
p53 蛋白表現量增加，因而影響下游基因的表現。我們的microarray
資料顯示，處理PsA 會明顯的改變人類子宮頸癌細胞中基因的表現，
有顯著差異的約有266 個基因。並由生物學統整分析這些基因的生物
功能及參與調控轉錄的網路。未來我們將進一步探討p53 蛋白的穩定
性對於PsA 調控細胞生長和基因表現的重要性，並且進一步研究它的
生物相關性。

An imbalance between histone deacetylases (HDAC) and histone
acetyltransferases (HAT) activities has been reported in cancer tissues or cells. HDAC is one of key players in tumorigenesis and angiogenesis. In recent years, inhibition of HDAC is a rapidly growing and very promising target for the development of anticancer drug, and it intended to reverse aberrant epigenetic changes associated with cancer. Psammaplin A (PsA), first identified in the Psammaplysilla sponge in 1987, is a natural bromotyrosine derivative. Recently, it was reported that PsA inhibits both HDAC and DNA methyltransferase (DNMT), as epigenetic modifiers of
tumor suppressor gene, and it has been suggested to be a promising novel HDAC inhibitor. However, it has not been tested in cervical cancer cells, and the accurate mechanism of PsA as a HDAC inhibitor is poorly understood. This study investigated the antitumor effect of PsA on human cervical cancer cells. We found that PsA significantly inhibited the
proliferation of HeLa cells in a dose-dependent manner. Additionally, it induced G1 phase arrest and apoptosis, and it can significantly up-regulate the mRNA and protein expression of p21 by altering p53 stability. We further discovered that PsA inhibited the expression of Mdm2 and decreased the interaction of p53 and Mdm2 by regulating
Mdm2 ubiquitination. Our microarray data showed that PsA treatment of HeLa cells significantly altered the gene expression profile, with about 266 genes undergoing upregulation and downregulation. Bioinformatic analysis revealed that these putative targets belong to diverse functional groups and transcriptional networks. In the future we will confirm the importance of p53 stability in PsA-mediated cell growth and gene expression control, and further investigate its biological relevance.

目錄
指導教授推薦書
口試委員會審定書
長庚大學授權書………………………………………………………...iii
致謝……………………………………………………………………...iv
中文摘要………………………………………………………………...vi
英文摘要……………………………………………………………….viii
目錄……………………………………………………………………….x
第一章 簡介……………………………………………………………1
1.1 組蛋白的修飾作用......................................................................1
1.1.1 組蛋白去乙醯酶.................................................................1
1.1.2 組蛋白去乙醯酶抑制劑.....................................................2
1.1.3 組蛋白去乙醯酶抑制劑結構分類.....................................3
1.2 Psammaplin A...............................................................................3
1.3 p53.................................................................................................5
1.3.1 調控p53 的穩定性.............................................................6
1.3.2 Mdm2 和p53 的降解..........................................................7
1.3.3 Mdm2 的功能......................................................................8
第二章 實驗材料與方法......................................................................10
xi
2.1 藥物來源....................................................................................10
2.2 細胞培養....................................................................................10
2.3 細胞增殖....................................................................................11
2.4 西方墨點法................................................................................11
2.5 細胞RNA抽取...........................................................................13
2.6 反轉錄-聚合酶連鎖反應..........................................................14
2.7 定量PCR....................................................................................14
2.8 細胞轉染....................................................................................15
2.9 Luciferease報導基因分析..........................................................15
2.10 細胞週期及細胞凋亡之分析..................................................16
2.11 微陣列晶片..............................................................................16
第三章 實驗結果與討論......................................................................18
3.1 Psammaplin A抑制人類子宮頸癌細胞的增殖.........................18
3.2 Psammaplin A增加細胞週期G1期的分佈，進而誘發細胞
亡.................................................................................................18
3.3 Psammaplin A 誘發人類子宮頸癌細胞中p21 和p53 的表
量..............................................................................................19
3.4 Psammaplin A經由p53 independent誘發人類骨髓瘤細胞中p21
的表現量.....................................................................................21
xii
3.5 Psammaplin A影響人類子宮頸癌細胞中p53蛋白的半衰期...21
3.6 Psammaplin A 降低p53 蛋白的泛素化....................................22
3.7 Psammaplin抑制Mdm2泛素黏合酶的活性..............................23
3.8 藉由microarray來分析Psammaplin A抑制細胞增殖所扮演的
生物角色.....................................................................................25
第四章 結論.............................................................................................27
第五章 參考文獻.....................................................................................30
第六章 圖示.............................................................................................35
第七章 表格.............................................................................................47
第八章 附錄.............................................................................................67
xiii
圖表目錄
圖一 HeLa細胞處理PsA後，抑制細胞生長狀況和引發凋亡的情
形...............................................................................................................35
圖二 PsA 誘發人類子宮頸癌細胞HeLa 內p53 和p21 mRNA 和蛋白
質表現以及p21 的轉錄性........................................................................38
圖三 PsA 誘發人類骨髓瘤細胞株Saos-2 內p21 mRNA 和蛋白質表
現...............................................................................................................40
圖四 PsA 影響p53 蛋白的穩定性..........................................................41
圖五 PsA 影響p53 的蛋白泛素化..........................................................42
圖六 PsA 抑制Mdm2蛋白的泛素化......................................................43
圖七 Microarray 經電腦分析擷取在兩組間有特別明顯表現差異的基
因，將這266 個基因利用PANTHER 分析其生物能...........................45
圖八 Microarray 中可能被p53 所調控的58 個基因，利用PANTHER
分析其生物功能.......................................................................................46
表一 PsA 改變HeLa 細胞的基因表現...................................................47
表二 Gene Go-轉錄調節分析.................................................................57
表三 p53 轉錄因子所調控的基因..........................................................59

1. Glozak MA, Sengupta N, Zhang X, Seto E: Acetylation and deacetylation of
non-histone proteins. Gene 2005, 363:15-23.
2. Pan LN, Lu J, Huang B: HDAC inhibitors: a potential new category of
anti-tumor agents. Cell Mol Immunol 2007, 4(5):337-343.
3. Feinberg AP, Ohlsson R, Henikoff S: The epigenetic progenitor origin of
human cancer. Nat Rev Genet 2006, 7(1):21-33.
4. Jenuwein T, Allis CD: Translating the histone code. Science 2001,
293(5532):1074-1080.
5. Cosgrove MS, Wolberger C: How does the histone code work? Biochem Cell
Biol 2005, 83(4):468-476.
6. Mutskov V, Felsenfeld G: Silencing of transgene transcription precedes
methylation of promoter DNA and histone H3 lysine 9. EMBO J 2004,
23(1):138-149.
7. Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G,
Bonaldi T, Haydon C, Ropero S, Petrie K et al: Loss of acetylation at Lys16
and trimethylation at Lys20 of histone H4 is a common hallmark of
human cancer. Nat Genet 2005, 37(4):391-400.
8. Wade PA: Transcriptional control at regulatory checkpoints by histone
deacetylases: molecular connections between cancer and chromatin. Hum
Mol Genet 2001, 10(7):693-698.
9. Yoshida M: [Potent and specific inhibition of mammalian histone
deacetylase both in vivo and in vitro by trichostatin A]. Tanpakushitsu
Kakusan Koso 2007, 52(13 Suppl):1788-1789.
10. Hong J, Ishihara K, Yamaki K, Hiraizumi K, Ohno T, Ahn JW, Zee O, Ohuchi
K: Apicidin, a histone deacetylase inhibitor, induces differentiation of
HL-60 cells. Cancer Lett 2003, 189(2):197-206.
11. Richon VM, Sandhoff TW, Rifkind RA, Marks PA: Histone deacetylase
inhibitor selectively induces p21WAF1 expression and gene-associated
histone acetylation. Proc Natl Acad Sci U S A 2000, 97(18):10014-10019.
12. Qiu L, Burgess A, Fairlie DP, Leonard H, Parsons PG, Gabrielli BG: Histone
deacetylase inhibitors trigger a G2 checkpoint in normal cells that is
defective in tumor cells. Mol Biol Cell 2000, 11(6):2069-2083.
13. Jung JH, Sim CJ, Lee CO: Cytotoxic compounds from a two-sponge
association. J Nat Prod 1995, 58(11):1722-1726.
14. Li CJ, Schmitz FJ, Kelly-Borges M: A new lysine derivative and new
3-bromopyrrole carboxylic acid derivative from two marine sponges. J Nat
Prod 1998, 61(3):387-389.
15. Shim JS, Lee HS, Shin J, Kwon HJ: Psammaplin A, a marine natural
product, inhibits aminopeptidase N and suppresses angiogenesis in vitro.
Cancer Lett 2004, 203(2):163-169.
16. Godert AM, Angelino N, Woloszynska-Read A, Morey SR, James SR, Karpf
AR, Sufrin JR: An improved synthesis of psammaplin A. Bioorg Med Chem
Lett 2006, 16(12):3330-3333.
17. Liu S, Fu X, Schmitz FJ, Kelly-Borges M: Psammaplysin F, a new
bromotyrosine derivative from a sponge, Aplysinella sp. J Nat Prod 1997,
60(6):614-615.
18. Park Y, Liu Y, Hong J, Lee CO, Cho H, Kim DK, Im KS, Jung JH: New
bromotyrosine derivatives from an association of two sponges, Jaspis
wondoensis and Poecillastra wondoensis. J Nat Prod 2003,
66(11):1495-1498.
19. Pina IC, Gautschi JT, Wang GY, Sanders ML, Schmitz FJ, France D,
Cornell-Kennon S, Sambucetti LC, Remiszewski SW, Perez LB et al:
Psammaplins from the sponge Pseudoceratina purpurea: inhibition of
both histone deacetylase and DNA methyltransferase. J Org Chem 2003,
68(10):3866-3873.
20. Tabudravu JN, Eijsink VG, Gooday GW, Jaspars M, Komander D, Legg M,
Synstad B, van Aalten DM: Psammaplin A, a chitinase inhibitor isolated
from the Fijian marine sponge Aplysinella rhax. Bioorg Med Chem 2002,
10(4):1123-1128.
21. Kim DH, Shin J, Kwon HJ: Psammaplin A is a natural prodrug that
inhibits class I histone deacetylase. Exp Mol Med 2007, 39(1):47-55.
22. Mora FD, Jones DK, Desai PV, Patny A, Avery MA, Feller DR, Smillie T,
Zhou YD, Nagle DG: Bioassay for the identification of natural
product-based activators of peroxisome proliferator-activated
receptor-gamma (PPARgamma): the marine sponge metabolite
psammaplin A activates PPARgamma and induces apoptosis in human
breast tumor cells. J Nat Prod 2006, 69(4):547-552.
23. Ahn MY, Jung JH, Na YJ, Kim HS: A natural histone deacetylase inhibitor,
Psammaplin A, induces cell cycle arrest and apoptosis in human
endometrial cancer cells. Gynecol Oncol 2008, 108(1):27-33.
24. Shangary S, Wang S: Targeting the MDM2-p53 interaction for cancer
therapy. Clin Cancer Res 2008, 14(17):5318-5324.
25. Attardi LD, Jacks T: The role of p53 in tumour suppression: lessons from
mouse models. Cell Mol Life Sci 1999, 55(1):48-63.
26. Evans SC, Lozano G: The Li-Fraumeni syndrome: an inherited
susceptibility to cancer. Mol Med Today 1997, 3(9):390-395.
27. Feki A, Irminger-Finger I: Mutational spectrum of p53 mutations in
primary breast and ovarian tumors. Crit Rev Oncol Hematol 2004,
52(2):103-116.
28. Kubbutat MH, Vousden KH: Keeping an old friend under control:
regulation of p53 stability. Mol Med Today 1998, 4(6):250-256.
29. Midgley CA, Lane DP: p53 protein stability in tumour cells is not
determined by mutation but is dependent on Mdm2 binding. Oncogene
1997, 15(10):1179-1189.
30. Varshavsky A: The ubiquitin system. Trends Biochem Sci 1997,
22(10):383-387.
31. Momand J, Jung D, Wilczynski S, Niland J: The MDM2 gene amplification
database. Nucleic Acids Res 1998, 26(15):3453-3459.
32. Wu X, Bayle JH, Olson D, Levine AJ: The p53-mdm-2 autoregulatory
feedback loop. Genes Dev 1993, 7(7A):1126-1132.
33. Marston NJ, Crook T, Vousden KH: Interaction of p53 with MDM2 is
independent of E6 and does not mediate wild type transformation
suppressor function. Oncogene 1994, 9(9):2707-2716.
34. Reinke V, Lozano G: The p53 targets mdm2 and Fas are not required as
mediators of apoptosis in vivo. Oncogene 1997, 15(13):1527-1534.
35. Momand J, Zambetti GP, Olson DC, George D, Levine AJ: The mdm-2
oncogene product forms a complex with the p53 protein and inhibits
p53-mediated transactivation. Cell 1992, 69(7):1237-1245.
36. Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, Vogelstein B:
Oncoprotein MDM2 conceals the activation domain of tumour suppressor
p53. Nature 1993, 362(6423):857-860.
37. Chene P: Inhibiting the p53-MDM2 interaction: an important target for
cancer therapy. Nat Rev Cancer 2003, 3(2):102-109.
38. Freedman DA, Levine AJ: Nuclear export is required for degradation of
endogenous p53 by MDM2 and human papillomavirus E6. Mol Cell Biol
1998, 18(12):7288-7293.
39. Haupt Y, Maya R, Kazaz A, Oren M: Mdm2 promotes the rapid degradation
of p53. Nature 1997, 387(6630):296-299.
40. Honda R, Tanaka H, Yasuda H: Oncoprotein MDM2 is a ubiquitin ligase E3
for tumor suppressor p53. FEBS Lett 1997, 420(1):25-27.
41. Kubbutat MH, Ludwig RL, Levine AJ, Vousden KH: Analysis of the
degradation function of Mdm2. Cell Growth Differ 1999, 10(2):87-92.
42. Tyers M, Willems AR: One ring to rule a superfamily of E3 ubiquitin
ligases. Science 1999, 284(5414):601, 603-604.
43. Roth J, Dobbelstein M, Freedman DA, Shenk T, Levine AJ:
Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels
of the p53 protein via a pathway used by the human immunodeficiency
virus rev protein. EMBO J 1998, 17(2):554-564.
44. Lain S, Midgley C, Sparks A, Lane EB, Lane DP: An inhibitor of nuclear
export activates the p53 response and induces the localization of HDM2
and p53 to U1A-positive nuclear bodies associated with the PODs. Exp
Cell Res 1999, 248(2):457-472.
45. Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM: A
leucine-rich nuclear export signal in the p53 tetramerization domain:
regulation of subcellular localization and p53 activity by NES masking.
EMBO J 1999, 18(6):1660-1672.
46. Tao W, Levine AJ: Nucleocytoplasmic shuttling of oncoprotein Hdm2 is
required for Hdm2-mediated degradation of p53. Proc Natl Acad Sci U S A
1999, 96(6):3077-3080.
47. Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, Murcia JM:
Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis.
Lesson from an uncleavable mutant. J Biol Chem 1998,
273(50):33533-33539.
48. Li LC, Carroll PR, Dahiya R: Epigenetic changes in prostate cancer:
implication for diagnosis and treatment. J Natl Cancer Inst 2005,
97(2):103-115.
49. Takai N, Desmond JC, Kumagai T, Gui D, Said JW, Whittaker S, Miyakawa I,
Koeffler HP: Histone deacetylase inhibitors have a profound antigrowth
activity in endometrial cancer cells. Clin Cancer Res 2004, 10(3):1141-1149.
50. Marks PA, Richon VM, Rifkind RA: Histone deacetylase inhibitors:
inducers of differentiation or apoptosis of transformed cells. J Natl Cancer
Inst 2000, 92(15):1210-1216.
51. Liu S, Bishop WR, Liu M: Differential effects of cell cycle regulatory
protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer
chemotherapy. Drug Resist Updat 2003, 6(4):183-195.
52. Ashcroft M, Vousden KH: Regulation of p53 stability. Oncogene 1999,
18(53):7637-7643.