Taiwanin E抑制檳榔鹼誘發口腔癌細胞轉移並透過ERK活化促進凋亡

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Title:	Taiwanin E抑制檳榔鹼誘發口腔癌細胞轉移並透過ERK活化促進凋亡 Effect of Taiwanin E on migration and apoptosis in arecoline/4-NQO-induced oral cancer cells via ERK activation
Authors:	劉珮潔;Pei-Jie Liu
Contributors:	醫學檢驗生物技術學系碩士班
Keywords:	Taiwanin E;口腔癌;轉移;促凋亡;Taiwanin E;oral cancer;migration;apoptosis
Date:	2013-08-14
Issue Date:	2013-10-02 11:27:33 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	口腔癌是一種高致命性的口腔疾病，必須早期診斷和治療。在台灣地區和部分的亞洲地區，嚼食檳榔非常盛行，而檳榔果實成分中的檳榔鹼具屬強力的致癌物質，慢性暴露於此致癌物質，可導致口腔黏膜上皮細胞的基因變化。中草藥具比較低的毒性，已普遍作為替代癌症的替代治療方式，本研究的主要目的是探討台灣杉萃取物Taiwanin E 對抗4-NQO和arecoline誘導之口腔癌細胞的分子機制探討。我們採用檳榔鹼和4-NQO誘導Narl mouse產生口腔癌T28細胞株，並與正常口腔N28細胞株作比較。當我們以臺灣杉天然萃取物Taiwanin E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；而N28細胞則無抑制效果及細胞毒性。Taiwanin E同時活化T28細胞中之p53、p21和p27蛋白大量表現，造成細胞週期調控蛋白Cyclin D1和Cyclin E的抑制，進而造成細胞G0/G1停滯。結果亦顯示Taiwanin E可減少β-catenin入核的現象，同時抑制癌細胞轉移。先前文獻指出β-catenin可能上調控到EGFR路徑,並活化Ras -Raf-MAPK 路徑，因此可作為一種新的癌症治療標的。結果證實亦Taiwanin E 僅可透過抑制p-ERK，但不影響到p-p38和p-JNK，來抑制T28。同時以在TUNEL及Flow 雙染分析證實，Taiwanin E並隨著劑量逐漸增加促進細胞凋亡，也可經由處理的時間增加促使T28細胞凋亡。此外，Taiwanin E透過抑制抗凋亡蛋白 Bcl-xL，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，促使細胞走向凋亡路徑。另進一步，我們採用ERK活化劑，證實Taiwanin E是否經抑制EPK，而促使細胞凋亡。結果中明顯看到抗凋亡蛋白Bcl-xL有明顯回復現象，而促凋亡蛋白Bax則減少顯著。總和以上結果，我們發現Taiwanin E 具有負調控 p-Tyr1068EGFR，抑制 p-ERK和β-catenin，進而抑制細胞增生和細胞轉移的能力，並活化 p53、p21和 p27，誘導細胞週期G0/G1停滯，同時活化促凋亡蛋白 Bax和釋放Cytochrome c和活化 Caspase-3，抑制抗凋亡蛋白 Bcl-2、p-Bad，進而促使口腔癌細胞凋亡。我們深信臺灣杉萃取物天然成份Taiwanin E可作為抑制檳榔鹼誘發之口腔癌增生及轉移並促使細胞走向凋亡。 Oral cancers can be life threatening if not diagnosed and treated early. Areca nut chewing is very popular in Taiwan and in other parts of Asia and chronic exposure to arecoline carcinogens in causes genetic changes in the epithelial cells of the oral mucosa. The use of herbs as alternative cancer therapies has attracted a great deal of attention owing to their lower toxicity. Therefore, the purpose of this study was to investigate the anti-cancer effect of Taiwanin E on arecoline and 4-NQO-induced oral cancer cell lines. 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 cells were cultured as T28 cancer cells. Taiwanin E used in this study was extracted from Taiwania cryptomerioides Hayata woud. T28 cells were treated with different concentrations of Taiwanin E and analyzed with MTT assay, western blot analysis, flow cytometry, TUNEL assay, and migration assay and their protein expression were analyzed by Western blotting. Taiwanin E significantly inhibited the cell viability of T28 cells in a dose dependent manner, but no cytotoxicity was observed in N28 normal cells. Taiwanin E activated p53, p21 and p27 proteins and reduced cell cycle regulatory proteins like Cyclin D1 and Cyclin E and thus resulted in G0/G1cell cycle arrest in T28 cells. Besides, Taiwanin E reduced the β-catenin translocation and inhibited migration ability of oral cancer. It had been reported that β-catenin is involved in EGFR pathways, and targets the Ras-Raf-MAPK pathways become a new therapy. The data show Taiwanin E regulated through p-ERK, it had not affect p-p38 and p-JNK. However,we used the annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining which showed that Taiwanin E strongly enhanced apoptosis in a dose-and time-dependent manner. Taiwanin E also decreased anti-apoptotic protein Bcl-xL and increased pro-apoptotic protein Bax, and down-regulated p-PI3K, p-AKT survival protein levels in T28 oral cancer cells. In addition, we applied the EPK activators to investigate whether p-ERK activated to inhibit cell apoptosis by Taiwanin E treated. The results indicated that Taiwanin E inhibited ERK and induced cell apoptosis. Taken together, Taiwanin E through the p-EGFR try1068, down-regulated the p-ERK, β-catenin protein expression, inhibited cell migration, and activated p21/p27 to induce G0/G1cell cycle arrest in T28 oral cancer cells. However, the p-EGFR try1068 also down-regulated the survival protein p-PI3K and p-Akt, decreased anti-apoptosis Bcl-xL, and increased pro-apoptosis Bax, released Cytochrome c from the mitochondria and induced Caspase family activation. We believe the Taiwanin E can apply as a potential treatment for arecoline-induced oral cancer.
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