臺灣杉萃取物拮抗口腔癌分子機制探討

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題名:	臺灣杉萃取物拮抗口腔癌分子機制探討 Molecular Mechanisms of Taiwania Extractive Component on Oral Cancer Cell Death
作者:	王舒懷;Wang, Shu-Huai
貢獻者:	基礎醫學研究所碩士班
關鍵詞:	臺灣杉;檳榔鹼;臺灣C;Taiwania cryptomerioides;arecoline;Taiwanin C
日期:	2012-08-20
上傳時間:	2012-09-25 14:11:02 (UTC+8)
出版者:	中國醫藥大學
摘要:	Part I 口腔癌是一種致命性的口腔疾病，嚼食檳榔在台灣地區非常盛行，檳榔果實中的檳榔鹼是一種強力的致癌物質，口腔黏膜長期接觸此致癌物質，易造成口腔上皮細胞基因體的改變。透過活化EGFR的活性使其下游基因COX-2過量表現造成上皮細胞的不正常增生，EGFR是一種生長因子的接受體，主要的功能為活化細胞的分化與增生。我們採用檳榔鹼和4-NQO誘導Narl mouse所產生的口腔癌T28細胞株與正常口腔N28細胞株作比較。當我們以臺灣杉天然萃取物Taiwanin C處理N28和T28細胞，T28細胞存活率隨著劑量增加而受到明顯抑制；N28細胞則無抑制效果及細胞毒性。Taiwanin C同時活化T28細胞中之p21、p27大量表現，並抑制細胞週期調控蛋白Cyclin A 、Cyclin B1、Cyclin D1和Cyclin E的含量,因而造成細胞G2/M停滯。除此之外，Taiwanin C同時透過抑制抗凋亡蛋白Bcl-2 、p-Bad表現量，增加促凋亡蛋白Bax的表現，以及負調控 p-PI3K、 p-Akt等細胞存活途徑蛋白表現，達到促使細胞凋亡。在TUNEL及Flow 雙染分析，更可見Taiwanin C隨著劑量增加逐漸促使T28細胞凋亡，Taiwanin C更明顯呈現抑制COX-2蛋白表現量和EGFR磷酸化蛋白的效果。總和以上結果，我們發現Taiwanin C 具有負調控p-Tyr1068EGFR/COX-2，活化p21、p27並誘導細胞週期G2/M停滯，同時活化促凋亡蛋白Bax及Caspase-3的表現，抑制抗凋亡蛋白Bcl-2、p-Bad，進而抑制初代培養的T28口腔癌細胞的不正常增生及促使口腔癌細胞的凋亡。我們相信臺灣杉萃取物Taiwanin C可作為抑制增生及促檳榔鹼誘發之口腔癌細胞凋亡之有效天然成份。 Part II 惡性腫瘤(癌症)是國人死因第一位，口腔癌已成為台灣所有癌症死亡原因的第五名，嚼食檳榔的習慣盛行於南亞，東南亞和台灣。檳榔鹼，為檳榔的主要生物鹼，已經有文獻指出在哺乳動物細胞會引起細胞毒性和遺傳毒性，甚至有助於致癌。我們採用檳榔鹼和4-NQO所誘導Narl mice產生的T28口腔癌細胞及N28正常口腔細胞。我們將N28和T28細胞處理臺灣杉中萃取出的天然產物Taiwanin C，T28細胞存活率隨著劑量增加而受到明顯抑制；而對N28正常口腔細胞則無細胞毒性的效果。我們進一步研究結果顯示，Taiwanin C亦能抑制T28細胞的爬行能力，同時抑制β-catenin及其下游細胞轉移的相關基因 Tbx3,c-Myc的蛋白表現量。同時，Taiwanin C抑制了β-catenin核內堆積的現象，主要透過促進β-catenin在proteasome的降解作用，並減低了β-catenin入核的蛋白量。而Taiwanin C主要是透過減少非活化態p-ser9 GSK-3β的蛋白含量，並增加活化態之GSK-3β，而造成β-catenin蛋白之降解。總和以上結果,我們發現Taiwanin C是透過活化GSK-3-β增加β-catenin的降解而減少β-catenin核內堆積並抑制其下游轉移相關基因的表現，進而阻斷口腔癌細胞T28轉移能力。相信Taiwanin C可作為抑制檳榔鹼誘導之口腔癌細胞轉移之天然藥物。 Part I Oral cancer is the major life-threatening oral diseases. Chewing Areca nut is a popular oral habit in Taiwan and Asia, arecoline is a potent carcinogen in Areca nut. Chronic exposure to arecoline carcinogens in the upper aerodigestive tract causes genetic changes in the epithelial cells of the oral mucosa. Arecoline may induce proliferative activity, through activate of the EGF receptor and promote downstream protein COX-2 over expression. The OSCC model in C57BL/6J Narl mouse is generated by 0.5 mg/mL arecoline plus 0.2mg/mL 4-NQO carcinogen in drinking water for 8 and 28 weeks to mimic the etiology of oral cancer patient in Asia. Mice were sacrificed and cell were cultured as T28 cancer cells. The treatment of natural herbal product from Taiwania cryptomerioides Hayata, Taiwanin C significantly inhibited the cell viability of T28 cells in a dose dependent manner, but no cytotoxicity effect on N28 normal cells. Taiwanin C activated p21 and p27 proteins and reduced the Cyclin A, Cyclin B1, Cyclin D1 and Cyclin E cell cycle regulatory proteins, which resulted in G2/M cell cycle arrest in T28 cells. Besides, by TUNEL assay and Flow-FITC measurement, Taiwanin C strongly enhanced T28 oral cancer cells apoptosis in a dose dependent manner. Taiwanin C also decreased anti-apoptotic protein Bcl-2 and p-Bad, increased pro-apoptotic protein Bax, and down-regulated p-PI3K, p-Akt survival protein levels in T28 oral cancer cells. Moreover, we observed that Taiwanin C inhibited p-Tyr1068EGFR and COX-2 protein expressions in T28 cells. Taken together, Taiwanin C down-regulated the p-Tyr1068EGFR/p-Akt signaling pathway to inhibit COX-2 expression and activated p21/p27 to induce G2/M cell cycle arrest, and also up regulated the expression of pro-apoptotic Bcl-2 family members to induce cell apoptosis, which resulted in the proliferative suppression and apoptosis promotion of T28 primary oral squamous cancer cells. We believe the Taiwanin C can apply as a potential treatment candidate for arecoline-induced oral cancer. Part II Cancer is the first leading cause of death, and cancer is the fifth leading cause of cancer-related death in Taiwan. Betel quid (Areca nut) chewing with areca nut is a habit seen throughout the world and wide spread habit in South, South-East Asia and Taiwan. Arecoline, the major alkaloid of areca nut, has been known to cause cytotoxicity and genotoxicity in mammalian cells and even contributes to carcinogenicity. The OSCC model in C57BL/6J Narl mice is generated by 0.5mg/ml arecoline plus 0.2mg/mL 4-NQO carcinogen in drinking water for 8 and 28 weeks to mimic the etiology of oral cancer patient in Asia. Mice were sacrificed and cell were cultured as T28 cancer cells. The treatment of nature herbal product from Taiwania cryptomerioides Hayata, Taiwanin C significantly inhibited the cell viability of T28 cells in a dose dependent manner, but no effect on N28 norrmal cells. Our results further revealed that Taiwanin C extract inhibited T28 cells migration ability in a dose dependent manner in wound healing and migration assays. Taiwanin C also reduced β-catenin protein level, down-regulated its downstream metastatic proteins, Tbx3 and c-Myc. Besides, Taiwanin C inhibited the nuclear accumulation and induced β-catenin degradation via proteasome-mediated pathway. Moreover, we found Taiwanin C enhanced GSK-3β and reduced the p-ser9 GSK-3β protein level to inactivate Wnt signaling and reduce β-catenin protein level. Taken together, we found that Taiwanin C blocked the metastasis effects of T28 cells mediated through GSK-3β activation to enhance protein degradation and reduce nuclear accumulation of β-catenin.
顯示於類別:	[基礎醫學研究所] 博碩士論文

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