Lovastatin 臨床上用於治療高血脂是利用其抑制mevalonate 的生合成路徑。抑制mevalonate 的生合成作用常引起抑制細胞增生和細胞凋亡。本篇論文主要研究 Lovastatin 是否會引發人類未分化甲狀腺癌細胞ARO 產生凋亡現象，進而瞭解其作用的分子機制。利用 DNA 片段化測定法與流式細胞分析法，我們發現 Lovastatin確實會引發 ARO細胞凋亡的發生，並具有濃度依賴性與處理時間依賴性。若ARO 細胞預先以cycloheximide 處理，將抑制Lovastatin誘發之凋亡作用並具濃度依賴性。這個數據顯示 Lovastatin 誘發之 ARO 細胞凋亡作用需要蛋白質的新生成。ARO 細胞處理 50 mM Lovastatin後誘發一連串的凋亡反應，包括 (依發生時間次序) cytochrome c由粒腺體釋出進入細胞質內，活化蛋白分解酵素caspase-2, -3和-9，以及 poly(ADP-ribose) polymerase (PARP) 的降解反應。然而，外加mevalonate 或 geranylgeraniol (GGOH) 則可以預防Lovastatin誘發的凋亡反應與 PARP 的降解反應發生，但farnesol (FOH) 卻無此效應。當ARO 細胞處理geranylgeranyltransferase (GGTase) 抑制劑 (GGTI-298) 阻斷geranylgeranylation作用時，亦會增加凋亡細胞的比率。這些數據暗示在Lovastatin 處理下，geranylgeranylation 作用對ARO 細胞存活的維持是必須的。為了支持這個觀點，我們證實 ARO 細胞處理 Lovastatin後，RhoA 和 Rac1之活化，由細胞質到細胞膜的易位反應強烈的被抑制 (Ras 卻無此效應)。再者，外加mevalonate 或 GGOH則可以預防 Lovastatin 對 RhoA 和 Rac1的易位反應抑制現象，外加FOH卻無此預防效應。綜合以上，我們的數據暗示，Lovastatin誘發的ARO細胞凋亡反應是透過抑制Rho家族蛋白的geranylgeranylation作用，而不是Ras家族蛋白的farnesylation作用。

Lovastatin has been used to treat hyperlipidemia through blocking the mevalonate biosynthesis pathway. Inhibition of mevalonate synthesis may result in anti-proliferation and cell apoptosis. The aim of the present study was to examine the apoptotic effect of lovastatin in human ARO cells and to delineate its molecular mechanism underlying. Using DNA fragmentation and flow cytometry analyses, we demonstrated that lovastatin induced the occurrence of apoptosis in ARO cells in a dose- and time-dependent manner. Pretreatment of ARO cells with cycloheximide (CHX) dose-dependently suppressed lovastatin-induced apoptosis, suggesting that de novo protein synthesis is required for lovastatin effect on the induction of apoptosis in ARO cells. Treatment of the cells with 50 mM lovastatin induced cytochrome c translocation from mitochondria to cytosol, increases in caspases 2, 3 and 9 activity, and poly (ADP-ribose) polymerase (PARP) degradation in a time-dependent manner. However, administration of mevalonate or geranylgeraniol (GGOH), but not farnesol (FOH), dose-dependently prevented lovastatin-induced PARP degradation and the occurrence of apoptosis, while treatment of ARO cells with geranylgeranyltransferase (GGTase) inhibitor, GGTI-298, which blocks the geranylgeranylation, induced an increase in the percentage of the apoptotic cells. These data suggest that geranylgeranylation is required for survival of the lovastatin-treated ARO cells. To support this notion, we demonstrate that treatment of the cells with lovastatin dose-dependently decreased the translocation of RhoA and Rac1, but not Ras, from cytosol to membrane fraction. Moreover, the inhibitions of lovastatin-induced membrane translocation in RhoA and Rac1 were prevented by mevalonate and GGOH, but not by FOH. In conclusion, our data suggest that lovastatin induced apoptosis in ARO cells by inhibiting protein geranylgeranylation of the Rho family, but not farnesylation of the Ras family.

目錄
內文標題 頁
第一章 緒言及文獻回顧........................................01
A. 癌症與治療................................................01
B. 甲狀腺癌..................................................03
C. 細胞凋亡..................................................05
D. 細胞分化與微絨毛結構......................................11
E. 癌細胞入侵/移動...........................................11
F. Statins與HMG-CoA reductase................................12
G. Statins與癌細胞...........................................14
H. Protein prenylation.......................................15
I. RAS 超級家族的活化........................................17
J. RAS 超級家族的功能........................................19
K. 本論文三大主題............................................23
J. 圖/表
圖1-1 細胞壞死與細胞凋亡的形態學特徵比較.....................07
圖1-2 Ras蛋白經過四道轉譯後修飾作用，使其具有細胞膜的轉位活性，並且得以執行其訊息傳遞的功能..................................16
圖1-3 Ras 的活化作用週期.....................................18
表1-1細胞凋亡與細胞壞死各種相異特質的綜合比較.................08
第二章 實驗材料與方法........................................25
A. 藥品與試劑................................................25
B. 抗體......................................................26
C. 細胞與細胞培養............................................26
D. 細胞生長度與存活度測試....................................26
E. 利用洋菜膠電泳技術分析DNA段裂.............................27
F. Caspases 活性分析 ........................................28
G. 利用流式細胞儀分析細胞凋亡................................29
(1) 細胞週期分析..............................................29
(2) Annexin V結合分析.........................................29
(3) Terminal transfer Utilizing Nick End Labeling (TUNEL) 染色測試............................................................30
H. 粒腺體內膜電位差變化之測定................................30
I. 細胞質溶液之抽取與細胞質內Cytochrome c 之測定.............31
J. 西方墨點法................................................31
K. 細胞全蛋白抽取 ...........................................32
(1) PARP 分解測試
(2) FAK與paxillin之磷酸化測試
L. 不溶性細胞微粒成份與細胞質部分之分離......................33
M. 電子顯微鏡檢查 ...........................................34
N. 甲狀腺球蛋白濃度測定......................................35
O. Matrigel 入侵試驗.........................................36
P. F-actin 染色與流式細胞儀分析..............................36
Q. 細胞附著力測試 ...........................................36
圖
圖 2-1 MTT測試法原理..........................................27
第三章 Lovastatin誘發人類甲狀腺未分化癌細胞凋亡，過程涉及抑制蛋白分子geranylgeranylation修飾作用與蛋白新合成反應.............37
A. 中文摘要..................................................38
B. 英文摘要 (ABSTRACT)........................................39
C. 序論.......................................................41
D. 結果............................................................43
1. Lovastatin 抑制ARO細胞生長.................................43
2. Lovastatin 誘發ARO細胞產生凋亡現象.........................43
3. Lovastatin 誘發ARO細胞產生凋亡現象涉及蛋白新合成作用.......44
4. Lovastatin 誘發ARO細胞產生凋亡過程涉及cytochrome c釋出與caspases活化..................................................45
5. 抑制ARO細胞之蛋白 geranylgeranylation 與 farnesylation作用皆會引起細胞凋亡發生............................................46
6. Geranylgeraniol可避免 Lovastatin 誘發之ARO細胞凋亡.........46
7. Lovastatin 抑制 Rho 家族蛋白由細胞質活化轉位至細胞膜之作用.47
8. RhoA and RhoA kinase 是維持ARO 細胞生存所必須的分子........48
E 討論.......................................................50
F. 圖
圖3-1 Lovastatin 之化學結構..................................40
圖3-2 Mevalonate 代謝路徑....................................40
圖3-3 Lovastatin 抑制ARO細胞生長.............................55
圖3-4 Lovastatin 誘發ARO細胞 DNA 片段化與 phosphatidylserine 外翻現象........................................................56
圖3-5 Lovastatin-誘發ARO細胞凋亡需涉及蛋白質新合成作用.......57
圖3-6 Lovastatin處理後，引發ARO細胞內粒腺體cytochrome c釋出，caspases之活化與PARP被切割分解................................58
圖3-7 Prenyltransferase抑制劑引發ARO細胞凋亡.................60
圖3-8 Mevalonate與GGOH處理能預防Lovastatin-誘發ARO細胞凋亡之發生............................................................61
圖3-9 Lovastatin 抑制RhoA 與 Rac1的細胞膜的易位反應..........62
圖3-10 阻斷RhoA/RhoA kinase訊息傳遞會引發ARO細胞凋亡之發生...63
圖3-11 Lovastatin 引發ARO甲狀腺未分化癌細胞凋亡發生之可能機轉............................................................64
第四章 Lovastatin,一種3-Hydroxy-3-methylglutaryl Coenzyme A Reductase 抑制劑, 誘發人類甲狀腺未分化癌細胞分化之反應 65
A. 中文摘要..................................................66
B. 英文摘要...................................................67
C. 序論.......................................................68
D. 結果.......................................................70
1. Lovastatin誘發ARO細胞產生微絨毛結構........................70
2. Lovastatin促使ARO細胞減少甲狀腺球蛋白......................70
E. 討論.......................................................71
F. 圖.........................................................73
圖4-1 Lovastatin誘發ARO細胞表面微絨毛結構之增加 (SEM)........73
圖4-2 Lovastatin誘發ARO細胞表面微絨毛結構與細胞質分泌型微粒之增加 (TEM)......................................................74
圖4-3 Lovastatin 刺激使ARO細胞甲狀腺球蛋白分泌大幅增加.......75
圖4-4 Lovastatin 誘發ARO甲狀腺未分化癌細胞之再分化可能機轉...76
第五章 Lovastatin抑制EGF-誘發人類甲狀腺未分化癌細胞之入侵性，過程涉及抑制蛋白分子geranylgeranylation修飾作用與RhoA/ROCK訊息傳遞途徑..........................................................77
A. 中文摘要...................................................78
B. 英文摘要 (ABSTRACT)........................................80
C. 序論.......................................................82
D. 結果.......................................................85
1. Lovastatin抑制ARO 甲狀腺未分化癌細胞的EGF-誘發細胞入侵性...85
2. Geranylgeraniol 與 mevalonate能減低lovastatin對ARO細胞入侵性的抑制性效....................................................85
3. 蛋白geranylgeranylation對ARO未分化癌細胞的EGF-誘發細胞入侵性是必須的......................................................86
4. Lovastatin抑制EGF-誘發之Rho GTPases由細胞質向細胞膜上易位結合的過程........................................................86
5. Rho/ROCK訊息傳遞途徑對ARO細胞的EGF-誘發細胞入侵性是必須的..87
6. Lovastatin藉抑制RhoA/ ROCK訊息傳遞途徑而降低ARO細胞-ECM的附著力............................................................88
7. Lovastatin 對ARO細胞型態及細胞骨骼重整的影響...............89
8. Lovastatin 抑制EGF-誘發之p125FAK/paxillin活化訊息途徑......89
E. 討論.......................................................91
F. 圖.........................................................95
圖5-1 Lovastatin抑制EGF-誘發穿透Matrigel覆膜的入侵性.........95
圖5-2 Geranylgeraniol 與 mevalonate 之處理能回復被lovastatin所抑制的ARO細胞入侵性...........................................97
圖5-3 蛋白geranylgeranylation修飾作用對EGF-誘發ARO細胞入侵性是必須的........................................................98
圖5-4 Lovastatin抑制EGF-誘發ARO細胞內Rho GTPases蛋白之易位至細胞膜的反應....................................................99
圖5-5 抑制RhoA/ROCK訊息傳遞途徑會減少EGF-誘發ARO細胞入侵性作用...........................................................100
圖5-6 Lovastatin藉抑制RhoA/ROCK訊息傳遞途徑之活化來減弱ARO細胞-ECM附著力 ...................................................101
圖5-7 Lovastatin對ARO細胞型態與EGF-誘發之actin stress fiber重整的影響.......................................................103
圖5-8 Lovastatin減少細胞內FAK蛋白量，也抑制EGF-誘發FAK and paxillin之磷酸化作用.........................................104
圖5-9 Lovastatin 抑制 ARO甲狀腺未分化癌細胞移動入侵性之可能機轉...........................................................105
第六章 總結與展望............................................106
參考文獻.....................................................109
附錄...........................................................125

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