臺灣博碩士論文加值系統

流行病學研究指出，臺灣地區口腔癌的發生與嚼檳榔有密切關係。 過去研究指出，以檳榔萃取物處理人類KB細胞株，會釋放出Cyclooxygenase (COX)-2及PGE2。我們在臺灣口腔癌中也發現有COX-2過度表現的現象。先前已有轉殖基因動物模式的研究證實,COX-2基因與大腸癌早期的癌化過程具有相關性,另外也有大量的相關證據表示COX-2的過度表現與腫瘤的血管新生以及轉移過程具有關聯。但同時也有其他研究顯示COX-2的表現可能引起p53表現,反而使細胞週期停止,抑制腫瘤的生長與形成。本研究擬探討COX-2在口腔癌進展過程所扮演的角色。首先我們構築數種不同的COX-2表現載體,並且將其以基因轉染方式送入人類口腔癌細胞SAS,並成功從SAS細胞中選殖出三株過度表現COX-2的細胞株, 命名為SAS/COX-2 c.2/5/6。 在小鼠腫瘤生長實驗中發現COX-2過度表現可促進口腔癌細胞SAS的腫瘤生長，過度表現COX-2腫瘤比轉殖對照載體的細胞株更具有促進血管新生的作用。西方墨點法結果分析顯示COX-2在口腔癌細胞可以誘導Bcl-2蛋白生成。在SAS細胞株添加PGE2，發現PGE2可以誘導Bcl-2蛋白生成。過度表現COX-2或外加PGE2 可以對抗 SAS細胞對 cisplatin 的凋亡反應。 PGE2 其訊息傳遞是經由細胞膜上的受體來傳達。我們發現COX-2的過度表現可以誘導EP2蛋白生成, 並藉由EP2 訊息傳導路徑引起Bcl-2表現量上升。此外本實驗也首次發現COX-2在口腔癌細胞的過度表現會引起Ferritin heavy chain蛋白的表現量增加。
由於本研究發現COX-2過度表現可促進口腔癌細胞SAS的腫瘤生長。 最近研究發現COX-2酶選擇性抑制celecoxib，可以幫助預防及治療結腸癌及肺癌，因此我們也以人類口腔癌細胞株SAS，CA9-22探討celecoxib對口腔癌細胞生長與增殖的影響，並進一步了解celecoxib誘導SAS cell apoptosis的可能機制。我們發現，celecoxib可以明顯抑制口腔癌細胞生長(p<0.05) ，且具有劑量依賴性 (dose-dependent) (IC50= 50 uM)。 藉由流式細胞儀分析細胞細胞週期結果顯示， celecoxib (50uM/24小時)可增加G1 phase細胞與sub G0細胞 (apoptotic cells)數目百分比。以西方墨點法發現， caspase 3的活化與PARP cleavage證明celecoxib可引起人類口腔癌細胞株SAS及CA9-22的細胞凋亡。 同時可觀察到DR5的上昇, celecoxib對細胞的凋亡可能經由extrinsic訊息傳導路徑而產生。

Cyclooxygenase-2 (COX-2) is the inducible form of the COX enzymes, it has been shown to overexpress in a variety of human malignancies. COX-2 catalyzes synthesis of prostaglandins (PGs) with diversified biological activities, and its dysregulation plays a vital role in inflammation, tissue damage, and tumorigenesis. Previously studies show that the COX-2 is involved in the early stages of the oncogenic process of colorectal tumors, and a great amount of evidence also suggests COX-2 upregulation is close associated with tumor invasion and metastasis. But some other reports point out that COX-2 overexpression may induce tumor suppressor protein p53 upregulation, and led to cell cycle arrest. Previous study showed that areca nut extract can induce COX-2 secretion in KB cells. The aim of this study is to investigate the possible role of COX-2 in oral carcinogenesis.
We transfected COX-2 expression vectors into human oral cancer SAS cells and successfully obtained clones with COX-2 overexpression (SAS/COX-2 c.2/5/6). Tumorigenesis assay showed that COX-2 transfected clones formed tumors in SCID mice more rapidly than the control cells. These tumors appeared to be highly vascularized using pathological examination. Immunoblot analysis showed that Bcl-2, an anti-apoptotic protein, was specifically up-regulated COX-2 transfectants but not in the control cells. The use of pharmacological inhibitors or activators revealed that prostaglandin EP(2) receptor but not other prostaglandin receptors is involved in COX-2-mediated Bcl-2 up-regulation. We also for the first time to show COX-2 can induce the expression of another anti-apoptotic protein ferritin heavy chain and the induction is through PGE2-independent manner. Furthermore, SAS/COX-2 cells, but not vector control cells, were resistant to cisplatin-induced cytotoxicity. These results suggest that overexpression of COX-2 enhances the tumorigenic activity of oral cancer cells by both suppressing apoptosis and actively promoting angiogenesis.
Celecoxib, a cyclooxygenase 2 inhibitor, has chemopreventive and therapeutic activities toward many epithelial malignancies. Celecoxib can induce apoptosis in various cancer cell lines through a mechanism that is independent of its cyclooxygenase 2 inhibitory activity but its effects on oral cancer cells is largely uncharacterized. We found celecoxib significantly inhibited the proliferation of SAS and CA9-22 oral cancer cell line in a dose-dependent manner with a 50% inhibition concentration (IC50) of celecoxib is 50uM. DNA flow cytometric analysis showed that Celecoxib treatment induces G1 phase arrest. Celecoxib treatment also caused significant apoptosis of SAS and CA9-22 cells as evidenced by Hoechst 33258 staining, DNA fragmentation and cleavage of PARP. Celecoxib treatment induced the expression of death receptors, particularly that of DR5. It seems celecoxib activates extrinsic apoptosis pathway.

Chapter I 7
INTRODUCTION 7
Oral squamous cell carcinoma 7
Oral carcinogenesis and tumor pregression 8
Cyclooxygenases (COXs) and prostaglandins (PGs) in human cancer 10
Prostaglandin signaling transduction pathways and EP/FP receptors 13
Apoptosis 19
Regulation of apoptosis 20
Bcl-2 gene family 25
TNF, TNF-related apoptosis-inducing ligand (TRAIL) and its receptors 28
Celecoxib 32
Research objectives 35
Chapter II 37
MATERIALS AND METHODS 37
Cell and cell culture 37
in vivo tumor model 37
Drug treatment 38
Cell viability (MTT) assay 38
Measurement of apoptosis by flow cytometry 39
RNA isolation and reverse transcriptase-PCR 40
Western blot analysis 41
H&E and immunohistochemistry analysis 43
Hoechst staining 44
Caspase inhibition 44
Cell death detection ELISA 45
Chapter III 46
RESULTS 46
COX-2 expression in oral cancer and stable line selection 46
COX-2 stimulation up-regulates Bcl-2 but not other Bcl-2 family members 47
Involvement of EP2 receptors in COX-2-mediated Bcl-2 up-regulation 48
COX-2 over-expression in oral cancer cell SAS induce tumor growth 48
COX-2 induce Ferritin heavy chain (FTH) expression in oral cancer cell SAS 50
Celecoxib inhibit oral cancer cell SAS and CA9-22 growth 51
Cell cycle analysis 51
Celecoxib treatment induced PARP cleavage and caspase activation 52
Celecoxib treatment induced DR5 expression 53
Celecoxib treatment up-regulated p53/p21/p27 and down-regulated cyclin D expression in oral cancer cell SAS and CA9-22 53
Chapter IV 74
DISSCUSION 74
FUTURE RESEARCHES AND PERSPECTIVES 82
Chapter V 84
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