臺灣博碩士論文加值系統

易斷裂的 (fragile) WWOX gene可表現出一個含雙色胺酸功能區氧化還原酶WW domain-containing oxidoreductase (亦稱之為WWOX、FOR或WOX1)之腫瘤抑制性蛋白質 (tumor suppressor)。在動物體內以及體外的實驗研究中皆發現，許多癌症細胞過度表現WOX1時會誘導細胞凋亡，也可以抑制腫瘤的生長。以往的研究發現WOX1 和其family proteins在分化程度較差的皮膚鱗狀上皮細胞癌 (cutaneous squamous cell carcinomas, SCC) 中表現量明顯減少，但是WWOX mRNA的表現量並沒有降低的現象，顯示WWOX mRNA轉譯成蛋白質的過程可能受到了阻礙。疣狀癌(Verrucous carcinoma)為鱗狀上皮細胞癌的一種特殊類型，先前的研究發現，持續性由動脈給予 (intra-arterial infusion) 一種葉酸拮抗劑methotrexate (MTX)來治療疣狀癌的病患時，在SCC的細胞中可以觀察到WOX1 蛋白質的表現量明顯增加，並且病患的腫瘤也有顯著消退的現象，顯示WOX1在癌症化學治療上可能具有某些角色。在本論文的研究中，我們證實MTX可誘導SCC-4和SCC-15細胞中caspases的活化並且造成細胞凋亡，但是在SCC-9細胞中卻無法造成這些現象。在對MTX感受度較高之細胞中發現，MTX所誘導的細胞死亡與SCC細胞內WOX1 mRNA以及蛋白質表現量增加有所關聯。此外，SCC-15細胞在給予MTX後，細胞內WOX1蛋白質在Tyr33和Tyr61這兩個胺基酸位置上的磷酸化有增加的情形。若是讓SCC-15細胞暫時性地表現一個可以抑制WOX1表現的siRNA，或著是dominant negative的WOX1時，則可以抑制由MTX所造成之細胞死亡。相對的，讓SCC-9細胞過度表現WOX1可以增強由MTX所誘導之死亡。 細胞自噬 (autophagy) 被認為是一種在養份不足的情況下，可以保護細胞以避免死亡的生存機制。有趣的是，SCC-15細胞在施予MTX後，和autophagy 有關的蛋白質LC3以及Beclin-1的表現量顯著減少，但其表現量在SCC-9細胞中則沒有明顯改變。MTX會誘導SCC-9細胞中autophagosome 形成初期極為重要之ATG5蛋白質的表現，但是MTX卻抑制了ATG5在SCC-15細胞中的表現量。 此外，MTX 刺激SCC-15細胞中mTOR pathway的活化, 然而卻抑制了SCC-9細胞中mTOR的活性。綜合上述研究可以得知，MTX可以藉由增加SCC細胞內WOX1的表現量來誘導細胞凋亡。從我們的研究結果推論，autophagy受到抑制的現象可能與MTX造成細胞凋亡有所關聯性。針對於MTX如何透過WOX1導致SCC 細胞死亡的分子機轉仍然在深入探討中。

Fragile WWOX gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase (designated WWOX, FOR or WOX1). Overexpressed WOX1 induces apoptosis in numerous cancer cells and suppresses tumor growth both in vitro and in vivo. Previous study showed significant reduction of WOX1 and its family proteins in poorly differentiated cutaneous squamous cell carcinomas (SCC) without down-regulation of WWOX mRNA, indicating a translational blockade of WWOX mRNA to protein. Verrucous carcinoma is a distinctive variant of SCC. Continuous intra-arterial infusion of methotrexate (MTX), a folate antagonist, significantly upregulates WOX1 protein expression in the SCC cells and results in complete tumor regression in patients with verrucous carcinoma. These observations imply a role of WOX1 in chemotherapeutic potential. In this study, we determined that MTX induced caspase activation and apoptotic death in SCC-4 and SCC-15, but not SCC-9 cells, in both time- and dose-dependent manners. MTX-induced cell death was associated with increased mRNA and protein levels of WOX1 in MTX-susceptible SCC cells. Phosphorylation of WOX1 protein at Tyr33 and Tyr61 was increased in SCC-15 cells following MTX treatment. Transient expression of an siRNA construct targeting WOX1 or a dominant negative in SCC-15 cells suppressed MTX-mediated cell death. Conversely, overexpression of WOX1 in SCC-9 cells enhanced death induced by MTX. Autophagy has been suggested to be a survival mechanism protecting cells from nutrient deprivation-induced death. Interestingly, the expression of autophagy-related proteins LC3 and Beclin-1 was significantly downregulated by MTX in SCC-15, but remained unchanged in SCC-9 cells. MTX induced protein expression of ATG5, which is essential for the early steps in autophagosome formation, in SCC-9 cells, but suppressed its expression in SCC-15 cells. Moreover, MTX stimulated the mTOR pathway in SCC-15 cells, whereas the mTOR activity was inhibited in SCC-9 cells following MTX treatment. Together, MTX induces apoptosis in SCC cells by upregulating WOX1. Our results suggest that the suppression of autophagy may be associated with MTX-mediated apoptosis. The molecular mechanism underlying MTX-mediated SCC cell death via WOX1 is being underway.

英文摘要 I
目錄 III
圖目錄 V
緒論
含雙色胺酸功能區氧化還原酶 1
WWOX和cancer的關連性 2
WWOX在細胞中的功能和角色 3
WWOX 在squamous cell carcinoma (SCC)中的角色 5
材料與方法
A 材料 7
A-1 細胞株 7
A-2 試劑藥品 7
A-3 抗體 9
A-4 耗材 10
A-5 儀器 11
B 方法 12
B-1 製備細胞培養液 12
B-2 細胞死亡之測定 13
B-3 4',6-diamidino-2-phenylindole (DAPI) 染色 14
B-4 Annexin V染色 15
B-5 大腸桿菌的轉形 (E. coli transformation) 16
B-6 質體DNA的製備 17
B-7 細胞轉形 18
B-8 萃取細胞蛋白 19
B-9 定量蛋白質濃度 20
B-10 聚丙烯醯胺膠體電泳 (SDS-PAGE) 和西方墨點法 (Western blotting) 21
B-11 反轉錄聚合酶連鎖反應 (RT-PCR) 24
B-12 DNA 電泳 (DNA electrophoresis) 27
實驗結果
1. 在MTX處理後SCC-4以及SCC-15會有細胞死亡的現象, 而SCC-9細胞則對MTX有極高的耐受性 28
2. MTX誘發SCC-15細胞的凋亡 28
3. MTX誘發SCC-15細胞凋亡時會誘導WOX1蛋白質的表現量增加， 並且進一步引發Caspase-9與Caspase-3的活化 29
4. MTX透過轉錄的層次誘導WOX1的表現量增加 30
5. WOX1參與在MTX誘發SCC細胞凋亡的途徑之中 30
6. WOX1的磷酸化參與在MTX誘發細胞凋亡的途徑之中 31
7. MTX影響SCC細胞內autophagy的現象 32
8. MTX會影響調控autophagy之mTOR訊息傳遞途徑的活化 33
討論
Autophagy與MTX誘導細胞凋亡之關係 35
SCC-9對於MTX的耐受性 36
MTX誘導細胞凋亡與WOX1表現的關係 37
WWOX與autophagy的關連性 38
總結 38
參考文獻 40
圖附錄 46

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