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系統識別號 U0026-0812200911374711
論文名稱(中文) 腫瘤細胞表現caspase-3會促進細胞移動及轉移能力
論文名稱(英文) Expression of Caspase-3 In Tumor Cells Promotes Cell Migration and Metastasis
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 93
學期 2
出版年 94
研究生(中文) 李建勳
研究生(英文) Chien-Hsun Li
電子信箱 monkeyuki2001@yahoo.com.tw
學號 s4692404
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 口試委員-張晃猷
指導教授-楊倍昌
口試委員-湯銘哲
口試委員-謝奇璋
口試委員-林以行
中文關鍵字 轉移  細胞移動 
英文關鍵字 metastasis  caspase-3  cell migration 
學科別分類
中文摘要   Caspase-3蛋白酶是一種細胞凋亡蛋白,它會裂解許多與細胞黏附,DNA修補及結構相關的蛋白。藉此造成細胞的不穩定而使細胞走向死亡。最近的研究顯示,caspase-3在器官發育及細胞分化上亦扮演了重要的角色。例如紅血球的成熟與骨骼細胞的分化。在臨床的觀察中發現,許多的腫瘤細胞會表現高量的caspase-3,其中尤以胃癌及子宮肌瘤為甚。然而,這些表現了caspase-3的癌細胞並未步向死亡,反而變得較為惡性化,其轉移能力及抗藥性也隨之增加。 我們認為caspase-3的過量表現可能會促進細胞的癌化。為了探討capspase-3是否會增加腫瘤細胞的惡性化,我們利用了MCF-7乳癌細胞來過量表現caspase-3,並在A549肺癌細胞株中使用RNA干擾的技術抑制caspase-3的生成。這些細胞在caspase-3的表現被改變了之後,並未影響到他們的死亡率及增殖能力,然而在MCF-7細胞中caspase-3的過量表現卻促進了細胞移動的能力。而它的兩個細胞黏附分子 FAK與paxillin也有隨之減少的情形。顯示這個細胞的黏附性較差,移動力便跟著增強。Caspase-3亦造成了MCF-7細胞中MMP-2的活化,可能會促進細胞的轉移能力。另一方面 A549 細胞的caspase-3被抑制後,細胞的移動能力就降低了。在分子層次的表現中,caspase-3的表現促進了Erk的磷酸化而降低了p38的活化。更進一步的探討發現,Erk的活化與caspase-3的活性沒有關係,應該是過量表現procaspase-3所造成的。在Erk的磷酸化被抑制之後,高量表現caspase-3的腫瘤細胞的移動能力就被顯著的降低了。顯示Erk的活化對於caspase-3所促進的細胞移動是很重要的。此外, 在裸鼠的肺臟癌細胞轉移試驗中, 我們亦發現caspase-3表現量較高的細胞會造成較嚴重的轉移。總而言之,caspase-3可能是藉由活化Erk及其下游基因的功能而增加癌細胞的移動能力及轉移能力。
英文摘要   Caspase-3 is a cysteine protease that primarily executes the death mission of cells by cleaving various substrates. In recent studies, caspase-3 plays roles in organ development, such as the maturation of erythrocytes. Caspase-3 overexpression was observed in numerous clinical cases including gastric carcinoma and leiomyoma. In addition, these tumor cells became more invasive and metastatic upon enhanced expression of caspase-3. We hypothesize that caspase-3 may promote tumorigenesis. To verify this presumption, caspase-3 was introduced into the MCF-7 breast cancer cell line, which was originally caspase-3 deficient. In parallel, the caspase-3 expression of A549 lung carcinoma cells was reduced by RNA interference (RNAi) strategy. MCF-7 cells overexpressing caspase-3 did not show significant traits of apoptosis. In addition, proliferation of the A549 cells transfected with caspase-3 siRNA was not different from their control groups. Overexpression of caspase-3 in MCF-7 cells reduced the expression of paxillin while the cells remained viable. In addition, the caspase-3 overexpressing MCF-7 cells possessed higher motility and secreted more MMP-2. On the contrary, transfection with caspase-3 siRNA reduced motility and invasiveness of A549 cells. In addition, by using the in vivo experimental lung metastasis model, we observed that caspase-3 increased the severity of metastasis. In both MCF-7 and A549 cells, we observed a caspase-3 mediated extracellular signal-regulated kinase (ERK) activation, which is independent of caspase-3 activity. In addition, Erk phosphorylation is dominant in caspase-3 mediated cell migration enhancement. Inhibition of Erk activation strongly decreased cell motility. On the other hand, the expression of caspase-3 downregulated phosphorylation of p38 MAP kinase. In conclusion, caspase-3 contributed to cell migration, invasiveness and metastasis through an ERK signalling-dependent pathway.
論文目次 Contents
Contents........................................................................................................................... I
Figure Index .................................................................................................................. III
 ................................................................................................................................ IV
Abstract ...........................................................................................................................V
Introduction .....................................................................................................................1

Materials & Methods .......................................................................................................6
Materials...................................................................................................................7
Methods ..................................................................................................................11
Plasmid construction .......................................................................................11
Cells and transfections.....................................................................................11
Western blot analysis .......................................................................................11
Gelatin zymography assay ...............................................................................12
Caspase-3 activity assay ...................................................................................12
Detection of apoptotic cells ..............................................................................13
Cell proliferation assay ....................................................................................13
Single cell motility assay..................................................................................13
Wound healing assay.......................................................................................14
Invasion assay..................................................................................................14
Experimental lung metastasis model ...............................................................14
Buffer Preparation .................................................................................................15

Results ............................................................................................................................20
Part I.......................................................................................................................21
Establishing Caspase-3 alteration systems ............................................................21
Tumor cells express different level of caspase-3..............................................22
Establishment of caspase-3 overexpressing system in MCF-7 cells.................22
RNAi-mediated suppression of caspase-3 inA549 cells....................................23
Dose-dependent RNAi-mediated inhibition of caspase-3 in MCF-7-C3 cells ..23
Stable caspase-3-altered tumor cell lines .........................................................24
Part II .....................................................................................................................25
Behaviors of The Caspase-3 Manipulated Tumor cells ........................................25
Alteration of caspase-3 expression does not affect basal level caspase-3 activity
of the tumor cells .............................................................................................26
RNAi of caspase-3 inhibitscisplatin-induced apoptosis in tumor cells.............26
Modification of expression level of caspase-3 did not affect cell viability........26
Altered expression of caspase-3 did not change cell proliferation...................26
Caspase-3 induces cell motility ........................................................................27
Caspase-3 induces MMP-2 activation in MCF-7-C3 cells and enhances
invasiveness of A549 cells................................................................................27
Caspase-3 enhances lung metastasis of A549-WT and MCF-7-C3 cells .........28
Part III ....................................................................................................................29
Molecular Mechanisms ..........................................................................................29
Caspase-3 regulates expression of paxillin and FAK ......................................30
Alteration of caspase-3 expression affected phosphorylation of MAP kinase .30
Caspase-3 induced Erk phosphorylation is independent of caspase-3 activity.31
Requirement for the Erk signaling pathway for caspase-3-induced cell
migration .........................................................................................................31

Discussion .......................................................................................................................32
References ......................................................................................................................36
Figures ............................................................................................................................40
Appendix ........................................................................................................................62
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